Synthesis of chirally purified substituted benzothiazole diamines

ABSTRACT

Methods for preparing chirally purified substituted 4,5,6,7-tetrahydro-benzothiazole diamines such as, for example, (6R)2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole and purifying a dominant enantiomer of substituted 4,5,6,7-tetrahydro-benzothiazole diamines from entantiomerically enriched mixtures of substituted 4,5,6,7-tetrahydro-benzothiazole diamines are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/049,235, filed Mar. 14, 2008, which claims priority to U.S.Provisional Appln. No. 60/894,829, filed Mar. 14, 2007 and U.S.Provisional Appln. No. 60/894,814, filed Mar. 14, 2007, all of which areincorporated herein by reference in their entireties.

GOVERNMENT INTERESTS

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PARTIES TO A JOINT RESEARCH AGREEMENT

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INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

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BACKGROUND

1. Field of Invention

Not applicable

2. Description of Related Art

The compound 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole isa synthetic aminobenzothiazole derivative whose (6S) enantiomer,commonly known as pramipexole and commercially available under theMirapex® name, is a potent dopamine agonist, and thus, mimics theeffects of the neurotransmitter dopamine. Pramipexole has also beenshown to have both neuroprotective and dopaminergic activities,presumably through inhibition of lipid peroxidation, normalization ofmitochondrial metabolism and/or detoxification of oxygen radicals.Therefore, pramipexole may have utility as an inhibitor of the celldeath cascades and loss of cell viability observed in neurodegenerativediseases and is indicated for treating Parkinson's disease, clusterheadaches, restless legs syndrome and bipolar disorder with only smalldaily doses required and tolerated by patients activates dopaminereceptors. Additionally, oxidative stress may be caused by an increasein oxygen and other free radicals, and has been associated with thefatal neurodegenerative disorder amyotrophic lateral sclerosis (ALS).ALS is a progressive neurodegenerative disorder involving the motorneurons of the cortex, brain stem, and spinal cord. About 10% of all ALSpatients are familial cases, of which 20% have mutations in thesuperoxide dismutase 1 (SOD-1) gene. The SOD-1 enzyme may play a pivotalrole in the pathogenesis and progression of familial amyotrophic lateralsclerosis (FALS). Recent studies also link the premature neuronal deathassociated with ALS to mutated mitochondrial genes which lead toabnormalities in functioning of the energy production pathways inmitochondria.

The neuroprotectant activity of both enantiomers of pramipexole havetypical therapeutic doses expected to be in the range of about 10 mg/dayto about 1,500 mg/day. However, the pramipexole's agonistic effect on ofthe D₂ family of dopamine receptors only requires therapeutic doses thatrange between 0.5 and 5.0 mg/day, and even these relatively low dosesadverse side effects have been reported. For example, the BoehringerIngelheim product insert for Mirapex® sets the maximally tolerated dosefor humans at 4.5 mg/day, and a dose of pramipexole as low as 1.5 mg hasbeen shown to cause somnolence in humans. Single dose toxicity ofpramipexole after oral administration has been studied in rodents, dogs,monkeys and humans. In rodents, death occurred at doses of 70-105 mg/kgand above which is equivalent to a human dose of 7-12 mg/kg/ orapproximately 500-850 mg for a 70 kg (−150 lb) individual. In dogs,vomiting occurred at 0.0007 mg/kg and above, while monkeys displayedmajor excitation at 3.5 mg/kg. In human subjects, an initial single doseof pramipexole of greater than 0.20 mg was not tolerated. All speciesshowed signs of toxicity related to exaggerated pharmacodynamicresponses to the pramipexole related to dopaminergic agonism.

Thus, a clinical use of pramipexole as a mitochondria-targetedantioxidant is unlikely, as the high doses needed for theneuroprotective or anti-oxidative/mitochondrial normalization action arenot accessible due to high dopamine receptor affinity associated withthe (6S) enantiomer. In contrast,(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine is aneffective mitochondria-targeted neuroprotectant that exhibits excellentanti-oxidative properties when administered without adverse sideeffects. Thus, higher doses of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine can betolerated by patients and will allow greater brain, spinal cord andmitochondrial concentrations increasing the degree to which oxidativestress and/or mitochondrial dysfunction may be reduced. Theneuroprotective effect of the compositions of this disclosure may alsobe derived at least in part from the ability of the (6R) enantiomer ofpramipexole to prevent neural cell death by at least one of threemechanisms. First, the (6R) enantiomer of pramipexole may be capable ofreducing the formation of reactive oxygen species in cells with impairedmitochondrial energy production. Second, the(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine maypartially restore the reduced mitochondrial membrane potential that iscorrelated with Alzheimer's, Parkinson's, Huntington's and amyotrophiclateral sclerosis diseases. Third, the (6R) enantiomer of pramipexolemay block the apoptotic cell death pathways which are produced bypharmacological models of Alzheimer's disease, Parkinson's disease,Huntington's disease, amyotrophic lateral sclerosis diseases andmitochondrial impairment. High doses of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine required toelicit these neuroprotective effects generally require highly purepreparations of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine which takeinto account the upper limit of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminecontamination (0.5 mg to 5.0 mg).

Processes for the preparation of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole using a simplealkylation reaction were first described in U.S. Pat. Nos. 4,843,086 and4,886,812. Other preparations of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole have beendescribed that involve reductive amination rather than simple alkylationand, thus, produce a mixture of the R and S optical isomers with nodirect means for further purification of the optical isomers. Theseknown processes for the production of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole are expensive,labor intensive, and utilize hydride reducing agents that pose safetyrisks. Furthermore, there are currently no known processes for thedirect synthesis of the pure(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine from adiamine. Therefore, the R isomer must be synthesized as a mixture of theoptical isomers that is purified by expensive and time consuming methodsthat may utilize other problematic substances. Moreover, known processesinvolving deamination result in a loss of enantiomeric purity, andmethods useful for resolution of the optical isomers from mixtures fallshort of producing chirally and chemically pure preparations of the Renantiomer or the S enantiomer.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the invention presented herein are directed to a processfor preparing a chirally purified substituted4,5,6,7,-tetrahydro-benzothaizole diamine including the steps of:heating a solution comprising entantiomerically enriched4,5,6,7-tetrahydro-benzothiazole diamine of general formula (I):

wherein:

R₁ represents a hydrogen atom, an alkyl group having 1 to 6 carbonatoms, an alkenyl or alkynyl group each having 3 to 6 carbon atoms, analkanoyl group having 1 to 6 carbon atoms, a phenyl alkyl or phenylalkanoyl group having 1 to 3 carbon atoms in the alkyl part, whilst theabove-mentioned phenyl nuclei may be substituted by 1 or 2 halogenatoms;

R₂ represents a hydrogen atom or an alkyl group with 1 to 4 carbonatoms;

R₃ represents a hydrogen atom, an alkyl group with 1 to 7 carbon atoms,a cycloalkyl group having 3 to 7 carbon atoms, an alkenyl or alkynylgroup having 3 to 6 carbon atoms, an alkanoyl group having 1 to 7 carbonatoms, a phenyl alkyl or phenyl alkanoyl group having 1 to 3 carbonatoms in the alkyl part, whilst the phenyl nucleus may be substituted byfluorine, chlorine or bromine atoms,

R₄ represents a hydrogen atom, an alkyl group with 1 to 4 carbon atoms,an alkenyl or alkynyl group having 3 to 6 carbon atoms; and

at least one or R₁, R₂, R₃ or R₄ is a hydrogen in an organic solvent;and an alkyl sulfonate or an alkyl halide in a solvent to form areaction mixture reacting the reaction mixture; and recovering achirally purified substituted 4,5,6,7-tetrahydro-benzothiazole diamine.

In various embodiments, the alkyl sulfonate may be of general formula(11):

wherein:

R′ is an alkyl group having 1 to 6 carbons, or a cycloalkyl, alkenyl,alkynyl, allyl, having 1 to 10 carbon atoms, or a benzyl, chlorobenzyl,phenyl or phenyl alkyl; and

Z is an alkyl group having 1 to 6 carbons, or a cycloalkyl, alkenyl,alkynyl, allyl, having 1 to 10 carbon atoms, or a benzyl, chlorobenzyl,phenyl or phenyl alkyl. In particular embodiments, X may be a propyland, in some embodiments, the alkyl sulfonate may be a propyl sulfonateselected from n-propyl tosylate, n-propyl methoxysulfonate andcombinations thereof. In other embodiments, the alkyl halide may be ofgeneral formula (12):

R′—X  (12)

wherein:

R′ is an alkyl group having 1 to 6 carbons, or a cycloalkyl, alkenyl,alkynyl, allyl, having 1 to 10 carbon atoms, or a benzyl, chlorobenzyl,phenyl or phenyl alkyl; and

X is any halide including, for example, fluorine, chlorine, bromine oriodide. In particular embodiments, X may be a propyl, and in someembodiments, the alkyl halide may be a propyl halide selected fromn-propyl bromide, n-propyl chloride, n-propyl fluoride, n-propyl iodideand combinations thereof.

The chirally purified substituted 4,5,6,7-tetrahydro-benzothiazolediamine of various embodiments may be at least greater than about 97%chirally pure. In some embodiments, the chirally purified substituted4,5,6,7-tetrahydro-benzothiazole diamine may be at least greater thanabout 99% chirally pure, and in other embodiments, chirally purifiedsubstituted 4,5,6,7-tetrahydro-benzothiazole diamine is at least about99.9% chirally pure.

The chemical purity of the substituted 4,5,6,7-tetrahydro-benzothiazolediamine of various embodiments may be greater than about 99%. In someembodiments, the chemical purity of the substituted4,5,6,7-tetrahydro-benzothiazole diamine may be greater than about99.9%, and in other embodiments, the chemical purity of the substituted4,5,6,7-tetrahydro-benzothiazole diamine is greater than about 99.99%.

In some embodiments, the entantiomerically enriched4,5,6,7-tetrahydro-benzothiazole diamine may be entantiomericallyenriched for an (6R) entantiomer and the chirally purified substituted4,5,6,7-tetrahydro-benzothiazole diamine is chirally purified for an(6R) entantiomer, and in particular embodiments, the entantiomericallyenriched 4,5,6,7-tetrahydro-benzothiazole diamine may beentantiomerically enriched for (6R)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole and the chirally purifiedsubstituted 4,5,6,7-tetrahydro-benzothiazole may be(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In otherembodiments, the entantiomerically enriched4,5,6,7-tetrahydro-benzothiazole diamine may be entantiomericallyenriched for an (6S)-entantiomer and the chirally purified substituted4,5,6,7-tetrahydro-benzothiazole diamine is chirally purified for an(6S) entantiomer, and in certain embodiments, the entantiomericallyenriched 4,5,6,7-tetrahydro-benzothiazole diamine may beentantiomerically enriched for (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole and the chirally purifiedsubstituted 4,5,6,7-tetrahydro-benzothiazole is(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In stillother embodiments, the entantiomerically enriched4,5,6,7-tetrahydrobenzothiazole diamine may be a ratio of greater thanabout 1:4 (6R)-entantiomer to (6S)-entantiomer to about 4:1(6R)-entantiomer to (6S)-entantiomer.

The solvent of various embodiments may be selected from an organicsolvent and an organic solvent mixed with water, and in someembodiments, the solvent may selected from, but not limited to, ethanol,1-propanol, n-butanol, dihydrofuran, dimethylformamide, dimethyl,dimethylacetamide, hexamethylphosphoric triamide or mixtures or hydratesthereof.

In some embodiments, the steps of heating, reacting and recovering eachindependently include stirring. In other embodiments, the steps ofheating and reacting may each independently be carried out at atemperature of from about 50° C. to about 125° C. In yet otherembodiments, the step of heating may further include adding the alkylsulfonate or alkyl halide to the heated 4,5,6,7-tetrahydro-benzothiazolediamine. In still other embodiments, the step of adding may be carriedout for about 0.5 hours to about 2 hours, and in certain embodiments,about 1.0 to about 2.0 molar equivalents of the alkly sulfonate or alkylhalide may be added. In particular embodiments, the step of reacting maybe carried out for up to about 12 hours. In yet other embodiments, thestep of recovering may include one or more steps selected from, but notlimited to, filtering the reaction mixture to isolate a precipitate,washing a precipitate, and drying a precipitate, and the process ofcertain embodiments may include the step of cooling the reaction mixtureto a temperature of about 25° C. after the step of reacting.

Other embodiments of the invention include a process for preparing achirally purified substituted 4,5,6,7-tetrahydro-benzothiazole diamineincluding the steps of heating a solution comprising anentantiomerically enriched 4,5,6,7-tetrahydro-benzothiazole diamine ofgeneral formula (1):

wherein:

-   -   R₁ represents a hydrogen atom, an alkyl group having 1 to 6        carbon atoms, an alkenyl or alkynyl group each having 3 to 6        carbon atoms, an alkanoyl group having 1 to 6 carbon atoms, a        phenyl alkyl or phenyl alkanoyl group having 1 to 3 carbon atoms        in the alkyl part, whilst the above-mentioned phenyl nuclei may        be substituted by 1 or 2 halogen atoms;    -   R₂ represents a hydrogen atom or an alkyl group with 1 to 4        carbon atoms;    -   R₃ represents a hydrogen atom, an alkyl group with 1 to 7 carbon        atoms, a cycloalkyl group having 3 to 7 carbon atoms, an alkenyl        or alkynyl group having 3 to 6 carbon atoms, an alkanoyl group        having 1 to 7 carbon atoms, a phenyl alkyl or phenyl alkanoyl        group having 1 to 3 carbon atoms in the alkyl part, whilst the        phenyl nucleus may be substituted by fluorine, chlorine or        bromine atoms,    -   R₄ represents a hydrogen atom, an alkyl group with 1 to 4 carbon        atoms, an alkenyl or alkynyl group having 3 to 6 carbon atoms;        and    -   at least one or R₁, R₂, R₃ or R₄ is a hydrogen in an organic        solvent;

adding to the heated solution propyl sulfonate or a propyl halide toform a reaction mixture; and

reacting the reaction mixture.

In some embodiments, the propyl sulfonate may be selected from n-propyltosylate, n-propyl methoxysulfonate and combinations thereof, and inother embodiments, the propyl halide is selected from n-propyl bromide,n-propyl chloride, n-propyl fluoride, n-propyl iodide and combinationsthereof.

The chirally purified substituted 4,5,6,7-tetrahydro-benzothiazolediamine of various embodiments may be at least greater than about 97%chirally pure. In some embodiments, the chirally purified substituted4,5,6,7-tetrahydro-benzothiazole diamine is at least greater than about99% chirally pure, and in other embodiments, the chirally purifiedsubstituted 4,5,6,7-tetrahydro-benzothiazole diamine is at least about99.9% chirally pure.

The chemical purity of the substituted 4,5,6,7-tetrahydro-benzothiazolediamine of various embodiments may be greater than about 99%. In someembodiments, the chemical purity of the substituted4,5,6,7-tetrahydro-benzothiazole diamine is greater than about 99.9%,and in other embodiments, the chemical purity of the substituted4,5,6,7-tetrahydro-benzothiazole diamine is greater than about 99.99%.

In certain embodiments, the entantiomerically enriched4,5,6,7-tetrahydro-benzothiazole may be enriched for(6R)4,5,6,7-tetrahydro-benzothiazole diamine and the chirally purifiedsubstituted 4,5,6,7-tetrahydro-benzothiazole diamine may be(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In certainother embodiments, the entantiomerically enriched4,5,6,7-tetrahydro-benzothiazole diamine may be enriched for (6S)4,5,6,7-tetrahydro-benzothiazole diamine and the chirally purifiedsubstituted 4,5,6,7-tetrahydro-benzothiazole diamine may be(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In someembodiments, the entantiomerically enriched4,5,6,7-tetrahydro-benzothiazole diamine may include a mixture of(6R)4,5,6,7-tetrahydro-benzothiazole diamine and(6S)4,5,6,7-tetrahydro-benzothiazole diamine and the chirally purifiedsubstituted 4,5,6,7-tetrahydro-benzothiazole diamine may include amixture of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamineand (6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. Inparticular embodiments, the mixture may be a racemic mixture. In otherembodiments, the enantiomerically enriched mixture may be a ratio ofgreater than about 1:4 (6R)4,5,6,7-tetrahydro-benzothiazole diamine and(6S)4,5,6,7-tetrahydro-benzothiazole diamine to about 4:1(6R)4,5,6,7-tetrahydro-benzothiazole diamine and(6S)4,5,6,7-tetrahydro-benzothiazole diamine.

In some embodiments, the solvent may be selected from a polar or organicsolvent and a polar or organic solvent mixed with water, and in certainembodiments, the solvent may be selected from ethanol, 1-propanol,n-butanol, dihydrofuran, dimethylformamide, dimethyl, dimethylacetamide,hexamethylphosphoric triamide or mixtures or hydrates thereof. In otherembodiments, the steps of heating and reacting each independently mayinclude stirring. In still other embodiments, the steps of heating,adding and reacting may each independently be carried out at atemperature of from about 50° C. to about 125° C. In yet otherembodiments, the process may further include the step of cooling thereaction mixture to a temperature of about 25° C. after the step ofreacting. In particular embodiments, the step of adding may be carriedout for up to about 2 hours. Various embodiments may further include thestep of recovering the chirally purified substituted4,5,6,7-tetrahydro-benzothiazole diamine, and in some embodiments,recovering may include one or more steps selected from filtering themixture to isolate a precipitate, washing a precipitate, and drying aprecipitate. In particular embodiments, about 1.0 to about 2.0 molarequivalents of the propyl sulfonate or propyl halide may be added.

Various embodiments of the invention also include chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole prepared by aprocess including the steps of: heating a solution comprisingentantiomerically enriched 2,6 diamino-4,5,6,7-tetrahydro-benzothiazoleand a propyl halide or a propyl sulfonate to form a reaction mixture;reacting the reaction mixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. In someembodiments, the propyl sulfonate may be selected, from, but not limitedto, n-propyl tosylate, n-propyl methoxysulfonate and combinationsthereof, and in other embodiments, the propyl halide may be selectedfrom n-propyl bromide, n-propyl chloride, n-propyl fluoride, n-propyliodide and combinations thereof.

In various embodiments, the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be at leastgreater than about 97% chirally pure. In some embodiments, the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may beat least greater than about 99% chirally pure, and in other embodiments,the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is at leastabout 99.9% chirally pure.

The chemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole of variousembodiments may be greater than about 99%. In some embodiments, thechemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is greater thanabout 99.9%, and in other embodiments, the chemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is greater thanabout 99.99%.

in particular embodiments, the entantiomerically enriched 2,6diamino-4,5,6,7-tetrahydro-benzothiazole may be a ratio of greater thanabout 2:1 (6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole to (6S)2,6diamino-4,5,6,7-tetrahydro-benzothiazole.

In some embodiments, the solvent may be selected from an organic solventand an organic solvent mixed with water, and in certain embodiments, thesolvent may be selected from ethanol, 1-propanol, n-butanol,dihydrofuran, dimethylformamide, dimethyl, dimethylacetamide,hexamethylphosphoric triamide or mixtures or hydrates thereof.

In various embodiments, the steps of heating and reacting may eachindependently include stirring, and in some embodiments, the steps ofheating and reacting may each be independently carried out at atemperature of from about 50° C. to about 125° C. In other embodiments,the process may further include cooling the reaction mixture to atemperature of about 25° C. after the step of reacting. In yet otherembodiments, the process may further include the step of adding thepropyl halide or propyl sulfonate to heated entantiomerically enriched2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, and in certainembodiments, the step of adding may be carried out for up to about 2hours. In particular embodiments, about 1.0 to about 2.0 molarequivalents of the propyl sulfonate or propyl halide may be added. Instill other embodiments, the step of reacting may be carried out for upto about 12 hours. In further embodiments, the step of recovering mayinclude one or more steps selected from filtering the mixture to isolatea precipitate, washing a precipitate, and drying a precipitate.

Various other embodiments of the invention include a process forpreparing a chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole including thesteps of heating a solution comprising 2,6diamino-4,5,6,7-tetrahydro-benzothiazole in an organic solvent; addingto the heated solution propyl sulfonate or a propyl halide to form areaction mixture; and reacting the reaction mixture for up to about 12hours. In some embodiments, the propyl sulfonate may be selected fromn-propyl tosylate, n-propyl methoxysulfonate and combinations thereof,and in other embodiments, the propyl halide is selected from n-propylbromide, n-propyl chloride, n-propyl fluoride, n-propyl iodide andcombinations thereof.

The process of embodiments may result in chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole that is at leastgreater than about 97% chirally pure. In some embodiments, the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may beat least greater than about 99% chirally pure, and in other embodiments,the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be at leastabout 99.9% chirally pure.

The chemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole of someembodiments may be greater than about 99%. In certain embodiments, thechemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be greaterthan about 99.9%, and in certain other embodiments, the chemical purityof the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may begreater than about 99.99%.

In some embodiments, the 2,6 diamino-4,5,6,7-tetrahydro-benzothiazolemay be (6R)2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In otherembodiments, the 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole may be(6S)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In stillother embodiments, the 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole maybe a mixture of (6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and(6S)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may bea mixture of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamineand (6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. Incertain embodiments, the mixture is a racemic mixture. In otherembodiments, the mixture may be a ratio of greater than about 1:4(6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole to about 4:1 (6R)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole and (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole.

The organic solvent of embodiments may be selected from an organicsolvent and an organic solvent is mixed with water, and in someembodiments, the organic solvent may be selected from ethanol,1-propanol, n-butanol, dihydrofuran, dimethylformamide, dimethyl,dimethylacetamide, hexamethylphosphoric triamide or mixtures or hydratesthereof.

In various embodiments, the steps of heating, adding and reacting eachindependently may include stirring, and in some embodiments, the stepsof heating, adding and reacting may each independently be carried out ata temperature of from about 50° C. to about 125° C. In certainembodiment, the process may further include the step of cooling thereaction mixture to a temperature of about 25° C. after the step ofreacting. In some embodiments, about 1.0 to about 2.0 molar equivalentsof the propyl sulfonate or propyl halide may be added, and the step ofadding in particular embodiments may be carried out for up to about 2hours. In still other embodiments, the process may include the step ofrecovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole, and in someembodiments, recovering may include one or more steps selected fromfiltering the mixture to isolate a precipitate, washing a precipitate,and drying a precipitate.

Embodiments of the invention further include a process for preparingchirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole including thesteps of heating a solution comprising 2,6diamino-4,5,6,7-tetrahydro-benzothiazole; adding a propyl halide or apropyl sulfonate to the heated solution slowly over from about 0.5 hoursto about 2 hours to form a reaction mixture; reacting the reactionmixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole.

In some embodiments, the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be at leastgreater than about 97% chirally pure. In other embodiments, the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may beat least greater than about 99% chirally pure, and in certainembodiments, the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is at leastabout 99.9% chirally pure.

The chemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole of variousembodiments may be greater than about 99%. In some embodiments, thechemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be greaterthan about 99.9%, and in particular embodiments, the chemical purity ofthe 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is greaterthan about 99.99%.

In some embodiments, the 2,6 diamino-4,5,6,7-tetrahydro-benzothiazolemay be (6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and thechirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In otherembodiments, the 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole is(6S)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In certainembodiments, the 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole may be amixture of (6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and(6S)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may bea mixture of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamineand (6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In somesuch embodiments, the mixture may be a racemic mixture. In other suchembodiments, the mixture may be a ratio of greater than about 1:4(6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole to about 4:1 (6R)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole and (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole.

In particular embodiments, the steps of heating, reacting and coolingeach independently may include stirring. In other embodiments, the stepof recovering may include cooling the mixture to a temperature of about25° C., and in still other embodiments, the step of recovering mayinclude stirring the reaction mixture for at least about 2 hours. In yetother embodiments, the step of recovering may further include one ormore steps selected from filtering the mixture to isolate a precipitate,washing a precipitate, and drying a precipitate. In various embodiments,the steps of heating, adding and reacting may each independently becarried out at a temperature of from about 50° C. to about 125° C., andin certain embodiments, the step of reacting may include stirring thereaction mixture for up to about 12 hours at from about 50° C. to about125° C.

Some embodiments of the invention include a chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole prepared by aprocess including the steps of heating a solution comprising 2,6diamino-4,5,6,7-tetrahydro-benzothiazole; adding a propyl halide or apropyl sulfonate to the heated solution slowly over from about 0.5 hoursto about 2 hours to form a reaction mixture; reacting the reactionmixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole.

In some embodiments, the propyl sulfonate is selected from n-propyltosylate, n-propyl methoxysulfonate and combinations thereof, and inother embodiments, the propyl halide is selected from n-propyl bromide,n-propyl chloride, n-propyl fluoride, n-propyl iodide and combinationsthereof.

In various embodiments, the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may at leastgreater than about 97% chirally pure. In some embodiments, the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may beat least greater than about 99% chirally pure, and in other embodiments,the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is at leastabout 99.9% chirally pure.

In various other embodiments, the chemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be greaterthan about 99%. In some embodiments, the chemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be greaterthan about 99.9%, and in other embodiments, the chemical purity of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole is greater thanabout 99.99%.

In some embodiments, the 2,6 diamino-4,5,6,7-tetrahydro-benzothiazolemay be a mixture of (6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazoleand (6S)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and the chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may bea mixture of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamineand (6S)2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. Incertain embodiments, the mixture may be a racemic mixture. In otherembodiments, the mixture may be a ratio of greater than about 4:1(6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole and (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole.

The organic solvent of various embodiments may be selected from aorganic solvent and an organic solvent is mixed with water, and theorganic solvent of some embodiments may be selected from ethanol,1-propanol, n-butanol, dihydrofuran, dimethylformamide, dimethyl,dimethylacetamide, hexamethylphosphoric triamide or mixtures or hydratesthereof.

In particular embodiments, the steps of heating, reacting and coolingeach independently may include stirring, and in some embodiments, thesteps of heating, adding and reacting are each independently carried outat a temperature of from about 50° C. to about 125° C. In otherembodiments, the process may further include cooling the reactionmixture to a temperature of about 25° C. after the step of reacting, andin still other embodiments, the step of adding is carried out for up toabout 2 hours. In certain embodiments, the step of recovering mayinclude one or more steps selected from filtering the mixture to isolatea precipitate, washing a precipitate, and drying a precipitate. Invarious embodiments, about 1.0 to about 2.0 molar equivalents of thepropyl sulfonate or propyl halide may be added.

Yet other embodiments of the invention include a process for preparingchirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole including thesteps of dissolving entantiomerically enriched2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in an organicsolvent to form a solution; heating the solution to from about 50° C. toabout 125° C.; adding an acid to the solution to form a reactionmixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole.

In some embodiments, about 1 molar equivalent to about 4 molarequivalents of the acid may be added. In other embodiments, recoveringmay include one or more steps including cooling the reaction mixture toa temperature of about 25° C.; stirring the reaction mixture for atleast about 2 hours; filtering the mixture to isolate a precipitate;washing a precipitate; and drying a precipitate.

Further embodiments of the invention include a chirally pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole prepared by sucha process.

Still other embodiments of the invention include a process for preparingchirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole including thesteps of dissolving entantiomerically enriched2-amino-4,5,6,7-tetmhydro-6-(propylamino)benzothiazole in an organicsolvent to form a solution; heating the solution to from about 50° C. toabout 125° C.; adding an achiral salt to the solution to form a reactionmixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole

In some embodiments, about 1 molar equivalent to about 4 molarequivalents of the achiral salt is added. In other embodiments, the stepof recovering may include one or more steps selected from cooling thereaction mixture to a temperature of about 25° C.; stirring the reactionmixture for at least about 2 hours; filtering the mixture to isolate aprecipitate; washing a precipitate; and drying a precipitate.

Further embodiments of the invention include a chirally pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole prepared by asuch a process.

Still further embodiments of the invention include a process forpreparing a 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazoledihydrochloride comprising dissolving a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt in anorganic solvent to form a solution; cooling the solution to atemperature of from about 0° C. to about 5° C.; adding concentrated HCland an organic solvent to the cooled solution; and stirring the solutionat a temperature of about 0° C. to about 5° C.

DESCRIPTION OF DRAWINGS

For a fuller understanding of the nature and advantages of the presentinvention, reference should be made to the following detaileddescription taken in connection with the accompanying drawings, inwhich:

FIG. 1A shows a reaction scheme illustrating the alkylation of4,5,6,7-tetrahydro-benzothiazole diamine.

FIG. 1B shows a reaction scheme illustrating the entantiomericpurification of one 4,5,6,7-tetrahydro-benzothiazole diamine from anentantiomeric mixture of 4,5,6,7-tetrahydro-benzothiazole diamines.

FIG. 2 shows a reaction scheme illustrating the preparation of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine-dihydrochlorideand(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine-difumerate.

FIG. 3A shows an exemplary HPLC trace of a mixture of (6R) and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine that isentantiomerically enriched for(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and acorresponding data table.

FIG. 3B shows an exemplary HPLC trace of chirally purified(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and acorresponding data table.

FIG. 4A shows an exemplary HPLC trace of Sample 118.

FIG. 4B shows an exemplary HPLC trace of Sample 105.

FIG. 4C shows an exemplary HPLC trace of Sample 061.

FIG. 4D show an exemplary HPLC trace of Sample 326A.

FIG. 5 shows an exemplary UV spectrum of a propyl tosylate peak elutedfrom an SPE column.

FIG. 6A shows an exemplary HPLC trace of a propyl tosylate standard.

FIG. 6B shows an exemplary HPLC trace of a(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine sample.

FIG. 7A shows an exemplary HPLC trace of a standard(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine.

FIG. 7B shows an exemplary HPLC trace of a sample(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, compositions, or methodologies described, as these may vary.It is also to be understood that the terminology used in the descriptionis for the purpose of describing the particular versions or embodimentsonly, and is not intended to limit the scope of the present inventionwhich will be limited only by the appended claims.

It must be noted that, as used herein, and in the appended claims, thesingular forms “a”, “an” and “the” include plural reference unless thecontext clearly dictates otherwise. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art. Although anymethods similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the present invention, thepreferred methods are now described. All publications and referencesmentioned herein are incorporated by reference. Nothing herein is to beconstrued as an admission that the invention is not entitled to antedatesuch disclosure by virtue of prior invention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Optional” or “optionally” may be taken to mean that the subsequentlydescribed structure, event or circumstance may or may not occur, andthat the description includes instances where the event occurs andinstances where it does not.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. “Administering” acomposition may be accomplished by oral administration, injection,infusion, absorption or by any method in combination with other knowntechniques. Such combination techniques include heating, radiation andultrasound.

The term “target”, as used herein, refers to the material for whicheither deactivation, rupture, disruption or destruction or preservation,maintenance, restoration or improvement of function or state is desired.For example, diseased cells, pathogens, or infectious material may beconsidered undesirable material in a diseased subject and may be atarget for therapy.

Generally speaking, the term “tissue” refers to any aggregation ofsimilarly specialized cells which are united in the performance of aparticular function.

The term “improves” is used to convey that the present invention changeseither the appearance, form, characteristics and/or physical attributesof the tissue to which it is being provided, applied or administered.“Improves” may also refer to the overall physical state of an individualto whom an active agent has been administered. For example, the overallphysical state of an individual may “improve” if one or more symptoms ofa neurodegenerative disorder are alleviated by administration of anactive agent.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate or prevent an unwanted condition or disease of apatient.

The terms “therapeutically effective amount” or “therapeutic dose” asused herein are interchangeable and may refer to the amount of an activeagent or pharmaceutical compound or composition that elicits abiological or medicinal response in a tissue, system, animal, individualor human that is being sought by a researcher, veterinarian, medicaldoctor or other clinician. A biological or medicinal response mayinclude, for example, one or more of the following: (1) preventing adisease, condition or disorder in an individual that may be predisposedto the disease, condition or disorder but does not yet experience ordisplay pathology or symptoms of the disease, condition or disorder, (2)inhibiting a disease, condition or disorder in an individual that isexperiencing or displaying the pathology or symptoms of the disease,condition or disorder or arresting further development of the pathologyand/or symptoms of the disease, condition or disorder, and (3)ameliorating a disease, condition or disorder in an individual that isexperiencing or exhibiting the pathology or symptoms of the disease,condition or disorder or reversing the pathology and/or symptomsexperienced or exhibited by the individual.

As used herein, the term “neuroprotectant” refers to any agent that mayprevent, ameliorate or slow the progression of neuronal degenerationand/or neuronal cell death.

The term “treating” may be taken to mean prophylaxis of a specificdisorder, disease or condition, alleviation of the symptoms associatedwith a specific disorder, disease or condition and/or prevention of thesymptoms associated with a specific disorder, disease or condition.

The term “patient” generally refers to any living organism to which tocompounds described herein are administered and may include, but is notlimited to, any non-human mammal, primate or human. Such “patients” mayor my not be exhibiting the signs, symptoms or pathology of theparticular diseased state.

As used herein, the terms “enantiomers”, “stereoisomers” and “opticalisomers” may be used interchangeably and refer to molecules whichcontain an asymmetric or chiral center and are mirror images of oneanother. Further, the terms “enantiomers”, “stereoisomers” or “opticalisomers” describe a molecule which, in a given configuration, cannot besuperimposed on, its mirror image.

As used herein, the terms “optically pure” or “entantiomerically pure”may be taken to indicate that a composition contains at least 99.95% ofa single optical isomer of a compound. The term “entantiomericallyenriched” may be taken to indicate that at least 51% of a composition isa single optical isomer or enantiomer. The term “entantiomericenrichment” as used herein refers to an increase in the amount of oneentantiomer as compared to the other. A “racemic” mixture is a mixtureof equal amounts of (6R) and (6S) enantiomers of a chiral molecule.

Throughout this disclosure, the word “pramipexole” will refer to (6S)enantiomer of 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazoleunless otherwise specified.

The term “trituration” may be taken to indicate a method of solidifyinga chemical compound. Trituration involves agitating the compound bystirring, beating or a method of the like until the chemical compoundforms a crystalline solid or precipitate. This solid may act to seed theremaining chemical compound in solution, causing it to precipitate orcrystallize from solution.

The term “pharmaceutical composition” shall mean a composition includingat least one active ingredient, whereby the composition is amenable toinvestigation for a specified, efficacious outcome in a mammal (forexample, without limitation, a human). Those of ordinary skill in theart will understand and appreciate the techniques appropriate fordetermining whether an active ingredient has a desired efficaciousoutcome based upon the needs of the artisan. A pharmaceuticalcomposition may, for example, contain(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine as theactive ingredient. Alternatively, a pharmaceutical composition maycontain (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or apharmaceutically acceptable salt of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine as theactive ingredient.

For the purposes of this disclosure, a “salt” is any acid addition salt,preferably a pharmaceutically acceptable acid addition salt, includingbut not limited to, halogenic acid salts such as hydrobromic,hydrochloric, hydrofluoric and hydroiodic acid salt; an inorganic acidsalt such as, for example, nitric, perchloric, sulfuric and phosphoricacid salt; an organic acid salt such as, for example, sulfonic acidsalts (methanesulfonic, trifluoromethan sulfonic, ethanesulfonic,benzenesulfonic or p-toluenesulfonic), acetic, malic, fumaric, succinic,citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic,oxalic and maleic acid salts; and an amino acid salt such as aspartic orglutamic acid salt. The acid addition salt may be a mono- or di-acidaddition salt, such as a di-hydrohalogenic, di-sulfuric, di-phosphoricor di-organic acid salt. In all cases, the acid addition salt is used asan achiral reagent which is not selected on the basis of any expected orknown preference for interaction with or precipitation of a specificoptical isomer of the products of this disclosure.

“Pharmaceutically acceptable salt” is meant to indicate those saltswhich are, within the scope of sound medical judgment, suitable for usein contact with the tissues of a patient without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al., (1977) J. Pharm.Sciences, Vol 6. 1-19, describes pharmaceutically acceptable salts indetail.

Embodiments of the invention described herein are generally directed toprocesses for the production of an entantiomerically and/or chemicallypure compound. More, specifically, embodiments of the invention aredirected to production of an entantiomerically and/or chemically purecompound using a trituration step where one enantiomer of anentantiomeric mixture of R and S stereoisomers of a compound isprecipitated out of solution and can be isolated by, for example, simplefiltering or other means for separating a solid or crystalline compoundfrom a solution.

For example, embodiments of the invention include a method forpreparation of an entantiomerically and chemically pure compound usingan one-pot bi-molecular nucleophilic substitution (S_(N)2) reactionsynthesis method as illustrated in the reaction scheme provided in FIG.1A. In FIG. 1A, a 4,5,6,7-tetrahydro-benzothiazole diamine (1) isreacted with an alkyl sulfonate (11) or a alkyl halide (12), exemplifiedby n-propyl sulfonate or propyl halide, to generate an aminoalkylcontaining compound of a 4,5,6,7-tetrahydro-benzothiazole diamine (2),and a sulfonate or halide salt. In a trituration step, one enantiomer ofthe 4,5,6,7-tetrahydro-benzothiazole diamine, for example,(R)(+)-4,5,6,7-tetrahydro-benzothiazole diamine (3), is precipitatedbased on insolubility of the enantiomers in the achiral halide orsulfonate salts produced as a result of the reaction and can beisolated. The other enantiomer remains in solution.

In another exemplary embodiment illustrated in FIG. 1B, chirally purecompounds, such as, (6R)4,5,6,7-tetrahydro-benzothiazole diamine (3),may be prepared from a mixture of R and S enantiomers of a compound,such as, for example, (6R)4,5,6,7-tetrahydro-benzothiazole diamine (4)and (6S)4,5,6,7-tetrahydro-benzothiazole diamine (5). In this process,trituration may result from the addition of an organic solvent and anachiral salt or acid to the mixture which may cause the formation of anacid addition salt of one enantiomer. The salt,(6R)4,5,6,7-tetrahydro-benzothiazole diamine (3), precipitates out ofthe solution based on insolubility of the enantiomers in the resultingsolution while the other enantiomer remains in solution. Theprecipitated crystalline enantiomer may then be isolated.

The reaction illustrated in FIG. 1A is not limited to a particular4,5,6,7-tetrahydro-benzothiazole diamine. For example, the4,5,6,7-tetrahydro-benzothiazole diamine may be any4,5,6,7-tetrahydro-benzothiazole diamine of formula (I):

wherein:

R₁ represents a hydrogen atom, an alkyl group having 1 to 6 carbonatoms, an alkenyl or alkynyl group each having 3 to 6 carbon atoms, analkanoyl group having 1 to 6 carbon atoms, a phenyl alkyl or phenylalkanoyl group having 1 to 3 carbon atoms in the alkyl part, whilst theabove-mentioned phenyl nuclei may be substituted by 1 or 2 halogenatoms;

R₂ represents a hydrogen atom or an alkyl group with 1 to 4 carbonatoms;

R₃ represents a hydrogen atom, an alkyl group with 1 to 7 carbon atoms,a cycloalkyl group having 3 to 7 carbon atoms, an alkenyl or alkynylgroup having 3 to 6 carbon atoms, an alkanoyl group having 1 to 7 carbonatoms, a phenyl alkyl or phenyl alkanoyl group having 1 to 3 carbonatoms in the alkyl part, whilst the phenyl nucleus may be substituted byfluorine, chlorine or bromine atoms,

R⁴ represents a hydrogen atom, an alkyl group with 1 to 4 carbon atoms,an alkenyl or alkynyl group having 3 to 6 carbon atoms;

and at least one or R₁, R₂, R₃ or R₄ is a hydrogen.

The 4,5,6,7-tetrahydro-benzothiazole diamine of formula 1 encompassesall enantiomers at any chiral center on the molecule.

The reaction illustrated in FIG. 1A is not limited by the type of alkylhalide or alkyl sulfonate utilized in the reaction. For example, thealkyl sulfonates may be any alkyl sulfonate of formula (II):

wherein:

R′ is an alkyl group having 1 to 6 carbons, or a cycloalkyl, alkenyl,alkynyl, allyl, having 1 to 10 carbon atoms, or a benzyl, chlorobenzyl,phenyl, phenyl alkyl and the like; and

Z is an alkyl group having 1 to 6 carbons, or a cycloalkyl, alkenyl,alkynyl, allyl, having 1 to 10 carbon atoms, or a benzyl, chlorobenzyl,phenyl, phenyl alkyl and the like; and the alkyl halide may be any alkylhalide of formula (12):

R′—X  (12)

wherein

R′ is an alkyl group having 1 to 6 carbons, or a cycloalkyl, alkenyl,alkynyl, allyl, having 1 to 10 carbon atoms, or a benzyl, chlorobenzyl,phenyl, phenyl alkyl and the like; and

X is any halide including, for example, chlorine, bromine or iodide. Invarious embodiments, R′ is an alkyl and, in particular embodiments, ann-propyl. In certain embodiments where alkyl sulfonate, the Z moiety maybe toluenesulfonate (tosylate) or methoxysulfonate. For example, invarious embodiments, the alkyl sulfonate may be n-propyl tosylate. Inembodiments in which an alkyl halide is used, the alkyl halide may ben-propyl bromide or n-propyl chloride. In general, the alkyl sulfonateor alkyl halide may be added in a quantity corresponding to about 1.0 toabout 4.0 molar equivalents of the diamine.

Advantages of embodiments such as those described above may include, forexample, (1) the use of simple reagents for the synthesis andpurification of one enantiomer enantiomeric compounds, (2) thesurprising improvement of the optical and chemical purity achieved bysimple trituration, and (3) such processes may be performed as a one-potsynthesis and purification reactions. When taken as a whole, theprocesses described above may be simpler, safer, and more efficient forthe production of chirally and chemically pure compounds. Additionally,the compounds may be sufficiently chirally and chemically pure to makesuch compounds safe an effective for use in pharmaceutical compounds andin the treatment of disease.

While the methods provided above may be used to purify any enantiomericcompound known in the art, particular embodiments of the invention aredirected to the production and optical purification of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole and, inparticular, (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine.The extremely high chiral and chemical purity of the compound producedby methods of the present invention allow for pharmaceuticalcompositions that may have a wide individual and daily dose range. Forexample, in some embodiments,(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine producedusing methods embodied herein may be nearly 100% chirally pure. Suchcompositions include little to no(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminecontamination and may be administered at high doses without the risk ofthe dopaminergic side effects associated with administration of highdose (6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. Thecompositions of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine maytherefore be used to treat neurodegenerative diseases, or thoseassociated with mitochondrial dysfunction or increased oxidative stresssuch as, for example, neurodegenerative dementias, neurodegenerativemovement disorders and ataxias, seizure disorders, motor neurondisorders or diseases, inflammatory demyelinating disorders and the likein adults and children. The compositions of this disclosure may also beuseful in the treatment of other disorders not listed herein, and anylisting provided in this disclosure is for exemplary purposes only andis non-limiting.

Compositions and pharmaceutical compositions comprisingentantiomerically pure(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine, such asthose discussed above, are further disclosed in U.S. application Ser.No. 11/773,642 entitled “Tetrahydrobenzothiazoles and Uses Thereof”filed Apr. 10, 2007, U.S. application Ser. No. 11/957,157 entitled“Compositions and Methods of Using R(+) Pramipexole”, filed Dec. 14,2007 and U.S. application Ser. No. 11/749,497 entitled “PramipexoleFormulations for the Treatment of Parkinson's Disease”, filed on May 16,2007, each of which are hereby incorporated by reference in theirentireties. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments of the present invention, the preferred methods, devices,and materials are described in detail herein.

Production of either enantiomer, (6R) or(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine may becarried using the bi-molecular nucleophilic substitution (S_(N)2)reaction described above and optical purification based on insolubilityof the compound product in achiral reagents. More specifically, FIG. 2schematically illustrates an embodiment of the process for theproduction of entantiomerically and chemically pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole using abi-molecular nucleophilic substitution (S_(N)2) reaction. In a firststep, 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole (7) is mixed propylp-toluenesulfonatc (propyl tosylate), and reacted to produce2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (8) and4-methylbenzenesulfonic acid (p-toluenesulfonic acid, p-TSA). Withoutwishing to be bound by theory, the diamine, 2,6diamino-4,5,6,7-tetrahydro-benzothiazole (7), in the reaction scheme ofFIG. 2 may act as the nucleophile in a nucleophilic attack on thesubstrate, a propyl tosylate, and the tosylate group may provide a goodleaving group as depicted below:

Thus, an embodiment of the invention is a process for preparing a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt (8) by abimolecular nucleophilic substitution reaction. In a second step, the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (8) may beentantiomerically purified by allowing the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (8) toprecipitate out of the reaction solution without the addition of anysecondary agents, such as, for example, additional salt. Thus,entantiomerically pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be isolatedin a third step by simply filtering out the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole crystals.

Some embodiments of methods of the invention may include additionalsteps. For example, in some embodiments, p-TSA may be removed to formthe entantiomerically purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (8) to form a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole free base (9)and hydrochloric acid or fumaric acid may be added to the free base toform2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole-dihydrochloride(14) or2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole-difumerate (15).In other embodiments,2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole-dihydrochloride(14) may be produced by adding hydrochloric acid to theentantiomerically purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (8).

While the reaction scheme illustrated in FIG. 2, shows the production of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine from(6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, in some embodiments,the same reaction may be carried out using (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole to produce(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. In otherembodiments, a mixture of (6R) and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine may be usedas starting material which may result in a mixture of (6R) and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. Inparticular embodiments, a mixture of (6R) and (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole that is entantiomericallyenriched for one entantiomer may be used as starting material.Additionally, the reaction illustrated in FIG. 2 may be carried out isan n-propyl halide or a mixture of n-propyl halide and n-propylsulfonate.

In some embodiments, step one of the process may include the additionalsteps of heating a diamine to form a solution or melt in a heating stepand adding the n-propyl halide or n-propyl sulfonate slowly over aperiod of time from, for example, about 0.5 hours to about 5 hours, inan additional step. In other embodiments, after the n-propyl halide orn-propyl sulfonate has been completely added to the 2,6diamino-4,5,6,7-tetrahydro-benzothiazole, the reaction may continueunder heating for an additional period of time ranging from, forexample, about 1 hour to about 12 hours, in a reaction step. In certainembodiments, the reaction mixture may be mixed by, for example, stirringfor one or more of the steps above or the reaction mixture may becontinually stirred from the heating step to the reaction step.Following the reaction step, the reaction mixture may be cooled and the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be isolatedand purified.

In embodiments such as those exemplified above, the diamine, 2,6diamino-4,5,6,7-tetrahydro-benzothiazole, may be a racemic mixture orentantiomerically enriched for either the S or R enantiomer, and the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole produced may begenerally optically enriched for the dominant enantiomer. For example,(6R) 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole may be the dominantenantiomer in mixtures used as a starting material in reaction thatproduces (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. Invarious embodiments, any R to S ratio of diamine may be used. Forexample, in some embodiments, the diamine may be in a racemic mixture(i.e., R:S is about 50:50), and in such embodiments, the yield of thereaction would be expected to be a racemic mixture of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine (i.e., about50:50). In other embodiments, the diamine may be provided in a mixturein which one stereoisomer is in excess over the other; for example, R:Smay be about 60:40. In such embodiments, the reaction would be expectedto yield a mixture of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine that isabout 60:40 R to S.

The reaction may be carried out in a melt or in a solvent or mixture ofsolvents, and the methods embodied herein are not limited by the type ornumber of solvents present during the reaction. Any solvent or mixtureof solvents known in the art in which the diamine and alkyl halide oralkyl sulfonate can dissolved may be used. For example, in variousembodiments, the solvents may be, for example, tetrahydrofuran,dimethylformamide, dimethly sulfoxide, dimethylacetamide,hexamethylohosphoric triamide, glacial acetic acid, pyridine, dioxan,ethanol, 1-propanol, i-propanol, n-butanol, i-butanol, or combinationsthereof, for example, dioxan/water, ethanol/water, tetrahydrofuran/waterand the like. In embodiments in which a combination of an organicsolvent and water are used, the organic solvent may have a water contentof from about 0 to about 10 volume percent. Preferably, the solventsused in the practice of this invention are standard ACS grade solvents.The selection of a solvent may enhance the reaction rate of the S_(N)2reaction. In some embodiments, one or more base such as, for example,sodium hydroxide, sodium hydride, potassium carbonate, sodium acetate,potassium-tertbutyloxide, triethylamine, di-isoproplyethylamine and thelike, may be additionally added to the reaction mixture which mayfurther enhance the efficiency of the reaction. When added, a base maybe present in a concentration of about 0.5 to about 3.0 equivalentsbased on the solvent. In still other embodiments, an alkylating agentmay be provided in the melt or solvent. Alkylating agents are well knownin the art and may be useful in embodiments of the invention. Forexample, alkylating agents may include, but not limited to,methyliodide, dimethylsulfate, ethylbromide, diethylsulfate,allyliodide, benzylbromide, 2-phenylethylbromidc andmethyl-p-toluenesulfonate.

In general, the reaction may be carried out under ambient conditions.However, the reaction temperature may vary among embodiments frombetween about −10° C. to about 50° C. and, in particular embodiments,from 0° C. to 30° C.

In further embodiments, dissolved diamine may be heated and mixed orstirred during the reaction. For example, various embodiments includethe step of heating a dissolved diamine, adding a n-propyl sulfonate orn-propyl halide which may, in some embodiments, be dissolved in asolvent to form a mixture, and stirring the mixture. In otherembodiments, a base such as di-isoproplyethylamine may be added to asolution including a diamine. N-propyl sulfonate or n-propyl halide maybe dissolved in a solvent, and then added to thediamine/di-isoproplyethylamine solution and this reaction mixture may bestirred. The temperature of reactions of such embodiments may;generally, be below the boiling temperature of the reaction mixture,more specifically, below the boiling temperature of the solvent(s) ofthe reaction mixture. For example, in some embodiments, an elevatedtemperature may be lower than about 125° C. In others, an elevatedtemperature may be lower than about 100° C., and in yet anotherembodiment lower than about 95° C., and in still others less than about75° C. Therefore, the reaction temperature may range from about 50° C.to about 125° C. in some embodiments, about 55° C. to about 100° C. inother embodiments, about 60° C. to about 95° C. in still otherembodiments, about 60° C. to about 75° C. in yet other embodiments andin certain embodiments, from about 55° C. to about 65° C.

The reaction time may vary within embodiments, and may depend upon, forexample, the identities of the reactants, the solvent system and thechosen temperature. For example, in some embodiments, the reaction timemay be from about 0.5 hours to about 12 hours. In other embodiments, thereaction time may be from about 1 hour to about 8 hours, and in certainembodiments, the reaction time may be about 4 hours. In general, thereaction time is chosen to provide sufficient time for substantially allof the diamine to undergo alkylation. In particular embodiments, thereaction time further provides sufficient time for the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole formed toprecipitate out of the reaction solution and from visible crystals.

Embodiments of the invention may further include the step of cooling thereaction to a temperature about room temperature (25° C.) following thereaction. In such embodiments, the reaction may be cooled for any amountof time with or without continued stirring. For example, in someembodiments, the reaction may be cooled with stirring for about 0.5 toabout 4 hours or more, and in other embodiments, the reaction may becooled with stirring for about 2 hours.

More specific embodiments may include the steps of: dissolving a diaminein dimethylformamide; heating the dissolved diamine to an elevatedtemperature; adding the n-propyl sulfonate or n-propyl halide dissolvedin dimethylformamide to form a reaction mixture; and stirring thereaction mixture for about 4 hours. In another embodiment, the steps mayinclude dissolving a diamine in dimethylformamide, heating the dissolveddiamine to an elevated temperature, slowly adding to the heateddissolved diamine 1.25 molar equivalents of n-propyl sulfonate orn-propyl halide dissolved in 10 volumes of dimethylformamide and 1.25molar equivalents of di-isoproplyethylamine with stirring the reactionover a period of about 4 hours. In yet another embodiment, 1.25 molarequivalents of di-isoproplyethylamine may be added to a diaminedissolved in dimethylformamide and the n-propyl sulfonate or n-propylhalide dissolved in dimethylformamide may be added to this mixture withstirring for about 4 hours.

Following synthesis, entantiomerically pure isomers of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be recoveredusing a trituration step in which the major isomer is isolated as aprecipitated crystals, while the minor stereoisomer remains in solution.Without wishing to be bound by theory, insolubility of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product inachiral reagents, such as p-TSA, may be independent of the R or Senantiomer, such that purity of the recovered isomer may depend only onthe volume of the reaction solution and starting percentage of the majorisomer. Thus, in an embodiment such as is described above, anentantiomerically pure(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine (R:S equals100:0) yield may be produced from a reaction in which diamine isprovided in an R to S ratio of 60:40.

An unexpected advantage of the process for preparing2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole described aboveis the limited solubility of the sulfonate or halide salt of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in polar organicsolvents which causes the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product toprecipitate once formed thereby purifying the final synthesis product,2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole, from thereaction mixture. In further embodiments, the substitution reaction suchas that illustrated in FIG. 2 may result in a sufficient achiral salt,p-TSA, concentration to cause the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole to becomeinsoluble and crystallize in the reaction solution without addingadditional agents, such as, for example, additional achiral salts.

The reaction embodied above and described in FIG. 2 may provide highlypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in aone-pot synthesis method. For example, in embodiments of the process,the chemical purity of the final2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole prepared may beat least 97%, 98%, and up to 100% without additional purification steps,and in particular embodiments, the chemical purity may be from 99.90% to100% without any additional purification steps. In still otherembodiments, the final2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may besubstantially free of achiral salts such as, for example, p-TSA. Forexample, in some embodiments, the achiral salt concentration of thefinal synthesis product may be less than 3%, and in others the achiralsalt concentration of the final synthesis product may be less than 1%,0.5%, 0.1%, 0.01%, 0.001% and so on. In certain embodiments, the achiralsalt concentration may be less than 1.5 ppm to less than 25 ppb or lessthan 0.00015% to less than 0.0000025%. Without wishing to be bound bytheory, the ability to produce highly chemically pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in a one-potmethod may demonstrate a significant advancement in the production of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. Moreover, suchpurity may provide pharmaceutical grade2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole more efficientlythan previous methods.

In various embodiments, the chiral purity for the R enantiomer preparedand purified may be greater than 99.6% about when a starting materialthat is entantiomerically enriched for R diamine is used. Similarly, invarious other embodiments, the chiral purity for the S enantiomerproduced and purified may be greater than 99.6% when a starting materialthat is entantiomerically enriched for S diamine is used. In someembodiments, the chiral purity for the R enantiomer prepared andpurified may be greater than 99.8% about when a starting material thatis entantiomerically enriched for R diamine is used. Similarly, in someother embodiments, the chiral purity for the S enantiomer produced andpurified may be greater than 99.8% when a starting material that isentantiomerically enriched for S diamine is used. In particularembodiments, the chiral purity for the R enantiomer prepared andpurified may be greater than 99.9% about when a starting material thatis entantiomerically enriched for R diamine is used. Similarly, inparticular other embodiments, the chiral purity for the S enantiomerproduced and purified may be greater than 99.9% when a starting materialthat is entantiomerically enriched for S diamine is used.

Not wishing to be bound by theory, the solubility of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine may be thesame in the trituration step of the synthesis and purificationprocesses. For example, if a synthesis process is carried out with 90grams of the (6R) diamine and 10 grams of the (6S)diamine, and thesolubility of the final2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product is 10grams for either enantiomer, then 80 grams of the(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine product and0 grams of the(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine productwould precipitate (assuming a 100% chemical conversion from the diamineand no change in molecular weight in going to the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product). Thatis, 10 grams of each enantiomer of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be expectedto go into solution. This would lead to a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product with a100% chiral purity for the (6R) enantiomer. The opposite ratio ofstarting materials for the synthesis process (90 grams of the (6S)diamine and 10 grams of the (6R) diamine) may generate a reactionproduct of 90 grams of the(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and 10 gramsof the (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. Fromthis reaction product mixture, 80 grams of the (6S) enantiomer and 0grams of the (6R) enantiomer of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole would beexpected to precipitate, leading to a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product with a100% chiral purity for the (6S) enantiomer. Thus, the volumes which areused for a reaction may have a large potential effect on the final yieldand chiral purity. That is, too large a volume will reduce the yield asmore of the 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazoleenantiomer products will go into solution (but increase the chiralpurity) and too small a volume will increase the yield as less of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole products will gointo solution (but reduce the chiral purity).

Other embodiments of the invention are directed to a process for thepurification of entantiomerically pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole from a mixtureof (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine including atrituration step. In some embodiments, the mixture of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine used in thepurification methods may be prepared as described herein above. In otherembodiments, the mixture of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine may beobtained using another method or form a commercially available mixtureof (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine.

In some embodiments, the trituration step may include the addition of anachiral salt to a solution containing a mixture of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. Asdescribed above, the addition of an achiral salt to a solutioncontaining a mixture of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine may causethe enantiomer having a greater concentration to become insoluble andform crystals in the solution. In some embodiments, the solutioncontaining a mixture of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine may beheated to an elevated temperature such as, for example, about 50° C. toabout 125° C., about 55° C. to about 100° C., about 60° C. to about 95°C. or about 60° C. to about 75° C. and an achiral salt may be added tothe solution. This solution may than be cooled from the elevatedtemperature to about room temperature slowly. For example in oneembodiment, the reaction may be cooled at a rate of about less than 25°C./hour. In another embodiment, the reaction may be slowly cooled andthe reaction solution may be stirred for at least about an additional 2hours. The rates of cooling and the time required for the additionalstirring may vary with the choice of achiral salt and may be easilyappreciated by one skilled in the art.

The crystalline(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine may then beisolated, washed and dried, and in various embodiments, may result inentantiomerically pure(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine having achemical purity of at least 97% and, in some embodiments, 98% to 100%.In such embodiments, the achiral salt may be any achiral salt listedhereinabove or any other achiral salt known in the art. Similarly, thesolvent of the(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine solution maybe any solvent described above in relation to the method for preparing2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. Either(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine or(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine may bepurified using the process of such embodiments. However, in certainembodiments, (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminemay be purified.

In other embodiments, entantiomerically enriched2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may betriturated from an acid addition solution based on the insolubility ofthe enantiomers in the achiral reagents. Various embodiments of thismethod may include the steps of dissolving an entantiomerically enriched2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in a solvent atan elevated temperature such as, for example, about 50° C. to about 125°C., about 55° C. to about 100° C., about 60° C. to about 95° C. or about60° C. to about 75° C., adding an acid to the dissolved2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole, cooling thereaction to about room temperature (25° C.) with stirring, stirring thecooled reaction mixture for an extended time at room temperature toallow formation of entantiomerically pure crystals and recoveringentantiomerically pure (6R) or(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine from thereaction mixture. In other embodiments, entantiomerically enriched2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be dissolvedin a solvent at an elevated temperature, about 0.5 equivalents to about2.05 equivalents of an acid may be added to the solution and thesolution may be cooled to room temperature. The cooled solution may thenbe stirred for an extended period of time and entantiomerically pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may berecovered. In particular embodiments, the selected acid may bep-toluenesulfonic acid (p-TSA) and the solvent may be ethanol. In otherembodiments, the temperature of the solution when the acid is added maybe lower than about 125° C., lower than about 100° C. or lower thanabout 75° C., and in certain embodiments, the temperature may be fromabout 65° C. to about 85° C. The cooling may generally occur slowly at,for example, a rate of about 25° C. per hour and the solution may bestirred for at least about 2 hours after 25° C. temperature has beenreached. The times necessary for the reaction may vary with theidentities of the reactants, the solvent system and with the chosentemperature, and may be easily appreciated by one of skill in the art.Reaction volumes may additionally dictate the degree of opticalpurification and the overall yield of the optically pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. These volumeswould be understood and appreciated by one of skill in the art. Examplesof specific times, temperatures and volumes which enable the practice ofthis invention are given in the Examples.

The solvent utilized may vary in embodiments and may generally be anorganic solvent such as, for example, acetonitrile, acetone, ethanol,ethyl acetate, methyl tert-butyl ether, methyl ethyl ketone, isopropylacetate, isopropyl alcohol and combinations thereof. In a particularembodiment, the organic solvent may be ethanol.

The acid of various embodiments may include: halogenic acids such as,for example, hydrobromic, hydrochloric, hydrofluoric and hydroiodicacid; inorganic acids such as, for example, nitric, perchloric, sulfuricand phosphoric acid; organic acids such as, for example, sulfonic acids(methanesulfonic, trifluoromethane sulfonic, ethanesulfonic,benzenesulfonic or p-toluenesulfonic), acetic, malic, fumaric, succinic,citric, benzoic, gluconic, lactic, mandelic, mucic, pamoic, pantothenic,oxalic and maleic acid; and amino acids such as aspartic or glutamicacid. The acid may be a mono- or di-acid, such as, for example, adi-hydrohalogenic, di-sulfuric, di-phosphoric or di-organic acid. Theacid of embodiments may be used as an achiral reagent which is,generally, not selected on the basis of any expected or known preferencefor interaction with, or precipitation of, a specific optical isomer ofthe 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole to beisolated. For example, in one embodiment, the selected acid may bep-toluenesulfonic acid. The amount of acid added may vary and isgenerally provided at about 1 molar equivalent to about 4 molarequivalents of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole.

Insoluble 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may beseparated from the reaction solution by any method known in the art. Forexample, in some embodiments, the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be collectedby simple filtering. There are numerous methods for filtering a solidfrom a solution, and any such method may by useful in embodiments of theinvention. In other embodiments, insoluble2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be isolatedby centrifugation. Again, such methods are well known in the art and anysuch method may be used in various embodiments of the invention. Theinsoluble crystalline2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may then bewashed to remove any contaminating solvent, sulfonate or halide salt, orsoluble enantiomer of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole from thecrystals using any method available. For example, in one embodiment, theprecipitated material may be washed in a volatile solvent such as analcohol or heptane followed by vacuum drying.

The 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole enantiomersprepared using methods above may be purified from a starting(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine mixture thatis enriched for one or the other enantiomers. For example, in someembodiments, the starting mixture may contain at least 55% or greater ofeither the R or S enantiomer, and in others the starting mixture maycontain about 70% or greater of either the R or the S enantiomer. Instill other embodiments, the starting material may contain greater thanabout 90% of either the R or S enantiomer. In particular embodiments,the starting mixture is enriched for(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine.

Without wishing to be bound by theory, the relative solubility of theoptical isomers of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in the achiralsalt or acid solutions allows for chiral and chemical purification thatis unexpected by using a relatively easy recovery method via a simpletrituration step. The enhanced enrichment resulting from thepurification methods described above result in that may reach opticalpurity. For example, in various embodiments, the final2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product may beenriched to 99% optical purity or greater, 99.5% optical purity orgreater, 99.8% optical purity or greater, and in certain embodiments,99.9% optical purity or greater. In still other embodiments, the opticalpurity of the final2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be 99.95% orgreater, or even 99.9.9% or greater. In particular embodiments, theoptical purity may be 100%.

The processes disclosed herein have several advantages. First, theprocesses avoid the use of borane reagents such as sodium borohydride,common in the reductive amination schemes used in the prior art, whichdecomposes rapidly to borane and hydrogen upon acidification. Second,reductive amination schemes involve the use of a two-step procedure inwhich the amide is formed first, followed by a reduction step. Themethods of this disclosure are one-pot synthesis and purificationprocedures, and therefore provide a safer, easier and more economicalsynthesis. Third, there is no loss of chirality during the alkylationprocesses of the S_(N)2 reaction mechanism of this disclosure, asopposed to previous synthesis schemes where chiral purity is oftenreduced or lost altogether during synthesis. Finally, the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product of thenucleophilic substitution reaction precipitates from the reactionmixture. This may be especially true for the p-TSA salt form of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. This is anunexpected advantage of the methods of the instant disclosure andprovides unique methods for chiral and chemical enrichment orpurification of the final2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product.

Additional embodiments of the invention include the conversion of eithersulfonate or halide salts of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole or free base2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole into anhydrochloric acid (HCl) salt as illustrated in FIG. 2. For example, insome embodiments, solid2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole sulfonate orhalide salt (8) may be re-dissolved in an alcohol, such as ethanol, andthe mixture may be cooled to between about 0 and about 5° C. withcontinuous stirring. Concentrated HCl may then be added, followed by asolvent such as methyl tert-butyl ether (MTBE), and the mixture may bestirred for about 0.5 to about 3 hours at between about 0 and about 5°C. until or until insoluble2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole dihydrochloridecrystals (10) have formed. The reaction mixture may then be filtered,washed in an inert solvent such as MTBE/alcohol solution and dried undervacuum. A detailed example of this synthesis may be found in Example 12.

In another embodiment, of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole sulfonate orhalide salts (8) may be converted to an HCl salt using a concentratedsolution of HCl and isopropyl acetate (IPAC). In such embodiments,2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole sulfonate orhalide salt (8) may be dissolved in IPAC and cooled to about 15° C. HCl(gas) may then be bubbled into the slurry for from about 0.5 hours to 3hours to produce 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazoledihydrochloride (10), after which the mixture may be filtered, washedwith an inert solvent, such as, for example, IPAC and dried under vacuumat room temperature. A detailed example of this synthesis may be foundin Example 13.

The sulfonate or halide salts of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole mayalternatively be converted to the free base form of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole as illustratedin FIG. 2. For example, in one embodiment, a p-TSA2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt (8) may bedissolved in dichloromethane (DCM) and water. The solution may then bybrought to a pH of about 11-12 using NaOH and resulting in the formationof two phases. The aqueous phase contains2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole that may beextracted with DCM, dried over magnesium sulfate (MgSO₄), filtered overCelite® and concentrated. The concentrated residue may be re-dissolvedin MTBE and stirred as a slurry for several hours. The solids may thenbe filtered, washed with MTBE, and dried under vacuum at a temperatureof about 35° C. The final product is2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole free base (9). Adetailed example of this synthesis may be found in Example 14.

In another embodiment, the sulfonate or halide salts of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (8) may beconverted to the free base form of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (9) bydissolving p-TSA salt of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in water andcooling the solution to a temperature of about 10° C. NaOH may be addedto the solution to increase the pH, the solution may be diluted andextracted several times in DCM. The combined organic phases are thenwashed, dried over MgSO₄, filtered and concentrated to dryness. Adetailed example of this synthesis may be found in Example 15.

In some embodiments, the free base form of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may be convertedto 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazoledihydrochloride (9) by bubbling HCl gas into a cooled solution of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole free base inIPAC. Alternatively, in other embodiments, the free base form of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (9) may beconverted to 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazoledihydrochloride (10) by mixing with concentrated HCl at room temperatureovernight. Detailed examples of such schemes may be found in Examples 16and 17, respectively. In still other embodiments, the free base form of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole (9) may beconverted to 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazolefumarate (11) by the addition of about 1 to about 4 molar equivalents offumaric acid.

The methods of the present disclosure require little time, utilizereadily available starting materials and do not involve the use ofhazardous or difficult to handle reagents. Each of the several steps ofthe methods disclosed as part of the present invention are high yieldingand afford products with very high chemical and chiral purity. Further,the processes disclosed herein may be scaled for industrial scalemanufacturing. As such, entantiomerically pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole may bemanufactured in batches of greater than 1 kg or more, 10 kg or more, oreven 25 kg or more as may be required to meet the needs of a large scalepharmaceutical use.

Embodiments of the invention also relate to a pure enantiomer of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole, either (6R) or(6S), produced by processes disclosed herein. Thus, an embodiment of theinvention is a chirally pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt prepared bya process which comprises dissolving 2,6diamino-4,5,6,7-tetrahydro-benzothiazole in an organic solvent, reactingthe 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole with a n-propylsulfonate or a n-propyl halide under conditions sufficient to generatethe 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt andrecovering the chirally pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt.

The present invention should not be considered limited to the particularembodiments described above, but rather should be understood to coverall aspects of the invention as fairly set out in the attached claims.Various modifications, equivalent processes, as well as numerousstructures to which the present invention may be applicable, will bereadily apparent to those skilled in the art to which the presentinvention is directed upon review of the present specification. Theclaims are intended to cover such modifications and devices. Theinvention and embodiments thereof illustrating the method and materialsused may be further understood by reference to the followingnon-limiting examples.

EXAMPLES

2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole was preparedusing the S_(N)2 substitution reaction described above. Reactions werecarried out under exemplary reactions conditions A, B and C describedbelow. Results of example syntheses using each of the several conditionswhich are embodiments of the present disclosure are listed in Table 1.Several example syntheses of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole using conditionsA, B and C of the present disclosure are detailed in Examples 1-5, theresults are presented in Table 1.

A: diamine dissolved in an organic solvent was heated to a reactiontemperature of less than about 125° C. with continuous stirring. Asolution of n-propyl sulfonate or n-propyl halide dissolved indi-isoproplyethylamine and an organic solvent was added to thedissolved, heated diamine slowly over a period of up to several hours toform a mixture, and this reaction mixture was stirred at the reactiontemperature for an additional period of time of up to about 4 hours.

B: diamine dissolved in an organic solvent was heated to a reactiontemperature of less than about 125° C. with continuous stirring. Asolution of n-propyl sulfonate or n-propyl halide dissolved indimethylformamide was added slowly over a period of up to several hoursto form a reaction mixture, and this reaction mixture was stirred at thereaction temperature for an additional amount of time up to about 4hours.

C: diamine was dissolved in dimethylformamide and heated to less thanabout 125° C. with continuous stirring. A solution of n-propyl sulfonateor n-propyl halide dissolved in dimethylformamide anddi-isopropylethylamine was added to the heated diamine slowly over aperiod of up to several hours to form a reaction mixture. This reactionmixture was then stirred at the reaction temperature for up to about 4hours.

Alternatively, di-isoproplyethylamine may be added to the heated diaminedissolved in an organic solvent prior to the addition of a solutionincluding n-propyl sulfonate or n-propyl halide dissolved indimethylformamide. As above, the n-propyl sulfonate or propylhalide/dimethlyformamide solution may be added slowly for a time periodup to several hours with continuous stirring and the reaction mixtureformed may be stirred at the reaction temperature for up to about 4hours.

These products were analyzed by high pressure liquid chromatography(HPLC) for chemical and chiral purity. ¹H NMR and ¹³C NMR was also usedto confirm the structure of the product is2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole

Example 1 Preparation of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition A

A 2.0 liter, three necked flask was equipped with an overhead stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 500 ml addition funnel. The flask was charged with 45grams of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine,followed by 750 ml of n-propanol. Under continuous stirring, the mixturewas heated to 95° C. over 15 minutes generating a clear solution. Theaddition funnel was charged with a solution of 74 grams propyl tosylateand 60 ml diisopropylethyleamine in 250 ml n-propanol. This solution wasadded dropwise to the 2.0 liter flask with continuous stirring over aperiod of 4 hours. The reaction was continued with stirring for anadditional 8 hours at 95° C., after which the solution was brought toroom temperature, and stirring was continued for an additional 4 hours.

The precipitated material was collected by filtration and washed threetimes using 100 ml reagent grade alcohol each time. The alcohol washedprecipitated cake was then washed with 100 ml heptane and dried underhigh vacuum for 2 hours.

The final weight of the dried product was 53.2 grams, representing a52.2% yield. HPLC was used to determine the chemical purity of the(6R)-2,6-diamino-4,5,6,7-tetrahydro-benzothiazole as 98.2% and thechiral purity as greater than 99.5%. ¹H NMR and ¹³C NMR were used toconfirm the structure.

Example 2 Preparation of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition A

A 250 ml, three necked flask was equipped with a magnetic stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 50 ml addition funnel. The flask was charged with 5grams of (6S)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followed by45 ml of n-propanol. Under continuous stirring, the mixture was heatedto a temperature of 95° C. over 15 minutes generating a clear solution.The addition funnel was charged with a solution of 8.2 grams propyltosylate and 6.7 ml diisopropylethyleamine in 16 ml n-propanol. Thissolution was added dropwise to the 250 ml flask with continuous stirringover a period of 2 hours. The reaction was continued with stirring foran additional 6 hours at 95° C., after which the solution was brought toroom temperature, and stirring was continued for an additional 4 hours.

The precipitated material was collected by filtration and washed threetimes using 10 ml reagent grade alcohol each time. The alcohol washedprecipitated cake was then washed with 10 ml heptane and dried underhigh vacuum for 2 hours.

The final weight of the dried product was 4.99 grams, representing a44.2% yield. HPLC was used to determine the chemical purity of the(6S)-2,6-diamino-4,5,6,7-tetrahydro-benzothiazole as 98.0% and thechiral purity as greater than 99.6%. ¹H NMR was used to confirm thestructure.

Example 3 Preparation of racemic2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole p-TSA salt:Condition A

A 250 ml, three necked flask was equipped with a magnetic stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 100 ml addition funnel. The flask was charged with 5grams of racemic 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followedby 80 ml of n-propanol. Under continuous stirring, the mixture washeated to a temperature of 95° C. over 15 minutes generating a clearsolution. The addition funnel was charged with a solution of 10.1 gramspropyl tosylate and 8.2 ml diisopropylethyleamine in 28 ml n-propanol.This solution was added dropwise to the 250 ml flask with continuousstirring over a period of 2 hours. The reaction was continued withstirring for an additional 6 hours at 95° C., after which the solutionwas brought to room temperature, and stirring was continued for anadditional 6 hours.

The precipitated material was collected by filtration and washed twotimes using 25 ml reagent grade alcohol each time. The alcohol washedprecipitated cake was then washed with 25 ml heptane and dried underhigh vacuum for 1 hour.

The final weight of the dried product was 5.12 grams, representing a 45%yield. HPLC was used to determine the chemical purity of the racemic2,6-diamino-4,5,6,7-tetrahydro-benzothiazole as 97.1%, and the chiralpurity showed a 1:1 mixture of the (6R) and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. ¹H NMR wasused to confirm the structure.

Example 4 Preparation of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition B

A 250 ml, three necked flask was equipped with a magnetic stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 50 ml addition funnel. The flask was charged with 5grams of (6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followed by50 ml of DMF. Under continuous stirring, the mixture was heated to atemperature of 75° C. 6.3 grams propyl tosylate was added dropwise tothe 250 ml flask with continuous stirring over a period of 6 hours.Progress of the reaction was monitored by analysis on HPLC.

The reaction was continued with stirring for an additional 12 hours atroom temperature. The solution was diluted with 20 ml. MTBE and stirredfor an additional hour. The precipitated material was collected byfiltration and washed with 20 ml MTBE, followed by 2 washes of 20 mleach ethanol. The washed precipitated cake was dried under high vacuum.

The final weight of the dried product was 4.6 grams, representing a 40%yield. HPLC was used to determine the chemical purity of the(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine as 94.9% andthe chiral purity as greater than 99.6%. ¹H NMR was used to confirm thestructure.

Example 5 Preparation of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition B

A 250 ml, three necked flask was equipped with a magnetic stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 50 ml addition funnel. The flask was charged with 10grams of (6S)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followed by100 ml of DMF. Under continuous stirring, the mixture was heated to atemperature of 75° C. The addition funnel was charged with a solution of16.4 grams propyl tosylate in 20 ml DMF. This solution was addeddropwise to the 250 ml flask with continuous stirring over a period of1.5 hours. Progress of the reaction was monitored by analysis on HPLC.

The reaction was continued with stirring for an additional 12 hours at75° C., after which the solution was brought to room temperature, andstirring was continued for an additional 7 hours. The solution wasdiluted with 100 ml MTBE and stirred for an additional hour. Theprecipitated material was collected by filtration and washed with 100 mlMTBE, followed by 2 washes of 50 ml each ethanol, and a wash with 50 mlheptane. The washed precipitated cake was dried under high vacuum.

The final weight of the dried product was 9.81 grams, representing a43.3% yield. HPLC was used to determine the chemical purity of the(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine as 99.4% andthe chiral purity as greater than 99.8%. ¹H NMR was used to confirm thestructure.

Example 6 Preparation of racemic2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole p-TSA salt:Condition B

A 250 ml, three necked flask was equipped with a magnetic stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 50 ml addition funnel. The flask was charged with 5grams of racemic-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followedby 50 ml of DMF. Under continuous stirring, the mixture was heated to atemperature of 75° C. 9.5 grams was added to the 250 ml flask withcontinuous stirring. Progress of the reaction was monitored by analysison HPLC.

The reaction was continued with stirring for an additional 4 hours at75° C., after which the solution was brought to room temperature, andstirring was continued for an additional 12 hours. The solution wasdiluted with 20 ml MTBE and stirred for an additional hour. Theprecipitated material was collected by filtration and washed with 50 mlMTBE, followed by 3 washes of 25 ml each ethanol, and the precipitatedcake was dried under high vacuum.

The final weight of the dried product was 2.9 grams, representing a25.6% yield. HPLC was used to determine the chemical purity of theracemic 2,6-diamino-4,5,6,7-tetrahydro-benzothiazole as 98.3%, and thechiral purity showed a 1:1 mixture of the (6R) and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. ¹H NMR wasused to confirm the structure.

Example 7 Preparation of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition C

A 12 L, three necked flask was equipped with an overhead stirrer, atemperature probe, a heating mantle, a claisen joint, a condenser, and a500 ml addition funnel. The flask was charged with 250 grams of (6R)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole, followed by 2 L of dimethylformamide (DMF). Under continuous stirring, the mixture was heated to atemperature of 65° C. The addition funnel was charged with a solution of386.6 grams propyl tosylate and 322 ml diisopropylethyleamine in 500 mlDMF. This solution was added to the 12 L flask dropwise over a period of2.0 hours. The reaction was monitored by analysis on HPLC.

The reaction was continued at 65° C. for an additional 5 hours, afterwhich the solution was gradually cooled to room temperature and stirredovernight. The solution was diluted with 2 L MTBE and stirred for anadditional 0.5 hours. The precipitated material was collected byfiltration and washed with 500 ml MTBE, followed by 3 washes of 500 mleach reagent alcohol. The washed precipitated cake was dried under highvacuum.

The final weight of the dried product was 317.6 grams, representing a56% yield. HPLC was used to determine the chemical purity of the(6R)-2,6-diamino-4,5,6,7-tetrahydro-benzothiazole as 98.4% and thechiral purity as greater than 99.8%. ¹H NMR and ¹³C NMR was used toconfirm the structure: ¹H NMR (300 MHz, DMSO-d6) δ 8.5 (br.s, 2H), 7.5(d, 2H), 71.2 (d, 1H), 6.8 (s, 2H), 3.4 (m, 1H), 2.95 (m, 3H), 2.6 (m,2H, merged with DMSO peak), 2.3 (s, 3H), 2.15 (m, 1H), 1.8 (m, 1H), 1.55(m, 2H), 0.9 (t, 3H); ¹³C NMR (300 MHz, DMSO-d6) δ 167.0, 145.5, 144.6,138.4, 128.6, 125.8, 110.7, 53.9, 46.5, 25.8, 25.6, 24.5, 21.2, 19.6,11.3.

Example 8 Preparation of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition C

A 500 ml, three necked flask was equipped with an overhead stirrer, atemperature probe, a heating mantle, a claisen joint, a condenser, and a100 ml addition funnel. The flask was charged with 20 grams of (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole, followed by 180 ml of dimethylformamide (DMF). Under continuous stirring, the mixture was heated to atemperature of 65° C. The addition funnel was charged with a solution of35.5 grams propyl tosylate and 32.8 ml diisopropylethylamine in 40 mlDMF. This solution was added to the 500 ml flask dropwise over a periodof 2.0 hours. The reaction was monitored by analysis on HPLC.

The reaction was continued at 65° C. for an additional 10 hours, afterwhich the solution was gradually cooled to room temperature and stirredfor 6 hours. The solution was diluted with 220 ml MTBE and stirred foran additional 0.5 hours. The precipitated material was collected byfiltration and washed with 50 ml MTBE, followed by 3 washes of 50 mleach reagent alcohol and a wash with 75 ml of heptane. The washedprecipitated cake was dried under high vacuum.

The final weight of the dried product was 25.4 grams, representing a 56%yield. HPLC was used to determine the chemical purity of the(6S)-2,6-diamino-4,5,6,7-tetrahydro-benzothiazole as 99.4% and thechiral purity as greater than 99.7%. ¹H NMR and ¹³C NMR was used toconfirm the structure: ¹H NMR (300 MHz, DMSO-d6) δ 8.5 (br.s, 2H), 7.5(d, 2H), 71.2 (d, 1H), 6.8 (s, 2H), 3.4 (m, 1H), 2.95 (m, 3H), 2.6 (m,2H, merged with DMSO peak), 2.3 (s, 3H), 2.15 (m, 1H), 1.8 (m, 1H), 1.55(m, 2H), 0.9 (t, 3H).

Example 9 Preparation of racemic2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole p-TSA salt:Condition C

A 250 ml, three necked flask was equipped with a magnetic stirrer, atemperature probe, a heating mantle, a claisen joint, a refluxcondenser, and a 50 ml addition funnel. The flask was charged with 5grams of racemic-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole, followedby 45 ml of DMF. Under continuous stirring, the mixture was heated to atemperature of 65° C. The addition funnel was charged with a solution of8.86 grams propyl tosylate and 8.2 ml of diisopropylethylamine in 10 ml.DMF. This solution was added dropwise to the 250 ml flask withcontinuous stirring over a period of 2 hours. Progress of the reactionwas monitored by analysis on HPLC.

The reaction was continued with stirring for an additional 6 hours at65° C., after which the solution was brought to room temperature. Thesolution was diluted with 70 ml MTBE and stirred for an additional hour.The precipitated material was collected by filtration and washed with 15ml MTBE, followed by 2 washes of 15 ml each ethanol, and a wash with 15ml heptane. The washed precipitated cake was dried under high vacuum.

The final weight of the dried product was 6.02 grams, representing a53.1% yield. HPLC was used to determine the chemical purity of theracemic 2,6-diamino-4,5,6,7-tetrahydro-benzothiazole as 99.2%, and thechiral purity showed a 1:1 mixture of the (6R) and(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. ¹H NMR wasused to confirm the structure.

Example 10 Preparation of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition E

A 1000 ml, three necked flask was equipped with an overhead stirrer, atemperature probe, a heating mantle, a claisen joint, a condenser, and a250 ml addition funnel. The flask was charged with 25 grams of (6R)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole, followed by 200 ml of dimethylformamide (DMF). Under continuous stirring, the mixture was heated to atemperature of 75° C. The addition funnel was charged with a solution of39.5 grams propyl tosylate and 32.5 ml diisopropylethyleamine in 50 mlDMF. This solution was added to the 1000 ml flask dropwise over a periodof 1.0 hours. The reaction was monitored by analysis on HPLC.

The reaction was continued at 75° C. for an additional 5 hours, afterwhich the solution was gradually cooled to room temperature and stirredovernight. The precipitated material was collected by filtration andwashed with 2 washes with 0.100 ml MTBE, followed by 3 washes of 75 mleach reagent alcohol and one wash with 125 ml of heptane. The washedprecipitated cake was dried under high vacuum.

The reaction resulted in a 47% yield. HPLC was used to determine thechiral purity as 99.8%. ¹H NMR and ¹³C NMR was used to confirm thestructure.

Example 11 Preparation of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition E

A 1000 ml, three necked flask was equipped with an overhead stirrer, atemperature probe, a heating mantle, a claisen joint, a condenser, and a250 ml addition funnel. The flask was charged with 25 grams of (6S)-2,6diamino-4,5,6,7-tetrahydro-benzothiazole, followed by 200 ml of dimethylformamide (DMF). Under continuous stirring, the mixture was heated to atemperature of 75° C. The addition funnel, was charged with a solutionof 39.5 grams propyl tosylate and 32.5 ml diisopropylethyleamine in 50ml DMF. This solution was added to the 1000 ml flask dropwise over aperiod of 2.0 hours. The reaction was monitored by analysis on HPLC.

The reaction was continued at 65° C. for an additional 5 hours, afterwhich the solution was gradually cooled to room temperature and stirredovernight. The precipitated material was collected by filtration andwashed with twice with 10 ml MTBE, followed by 3 washes of 75 ml eachreagent alcohol and one wash with 125 ml of heptane. The washedprecipitated cake was dried under high vacuum.

The reaction resulted in a 47% yield. HPLC was used to determine thechiral purity as 99.8%. ¹H NMR and ¹³C NMR was used to confirm thestructure.

TABLE 1 Experiments for S_(N)2 preparation of2-amino-4,5,6,7-tetrahydro-6- (propylamino)benzothiazole pTSA saltCondition Isomer Batch Size Results Ex. 1 (6R) 45 grams Yield = 53.2grams (52%) Chemical Purity = 98.2% AUC by HPLC Chiral Purity = >99.5%AUC by HPLC Ex. 2 (6S) 5 grams Yield = 4.99 grams (44.2%) ChemicalPurity = 98.0% AUC by HPLC Chiral Purity = >99.6% AUC by HPLC Ex. 3Racemic 5 gram Yield = 5.12 grams (45%) Chemical Purity = 97.1% AUC byHPLC Chiral Purity = 1:1 (6R):(6S) by HPLC Ex. 4 (6R) 5 gram Yield = 4.6grams (40%) Chemical Purity = 94.9% AUC by HPLC Chiral Purity = 99.6%AUC by HPLC Ex. 5 (6S) 10 gram Yield = 9.81 grams (43.3%) ChemicalPurity = 94.9% AUC by HPLC Chiral Purity = 99.7% AUC by HPLC Ex. 6Racemic 5 gram Yield = 2.9 grams (25.6%) Chemical Purity = 98.3% AUC byHPLC Chiral Purity = 1:1 (6R):(6S) by HPLC Ex. 7 (6R) 250 gram Yield =317.6 grams (56%) Chemical Purity = 99.4% AUC by HPLC Chiral Purity =99.8% AUC by HPLC Ex. 8 (6S) 20 gram Yield = 25.41 grams (56%) ChemicalPurity = 99.4% AUC by HPLC Chiral Purity = 99.7% AUC by HPLC Ex. 9Racemic 5 gram Yield = 6.02 grams (53.1%) Chemical Purity = 99.2% AUC byHPLC Chiral Purity = 1:1 (6R):(6S) by HPLC Ex. 10 (6R) 25 gram Yield =47% Chiral Purity = 99.8% AUC by HPLC Ex. 11 (6S) 25 gram Yield = 47%Chiral Purity = 99.8% AUC by HPLC

Example 11

Various ratios of entantiomerically enriched mixtures of (6R)2,6diamino-4,5,6,7-tetrahydro-benzothiazole and (6S)2,6diamino-4,5,6,7-tetrahydro-benzothiazole having (6R) to (6S) ratios of80:20, 20:80, 85:15, 15:85, 90:10, 10:90, 95:5 and 5:95, were used toprepare (6R) or(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine based on theenriched species in the starting material. The following reactionconditions were used:

F: 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole mixtures were dissolvedin 10 volumes of DMF and 1.25 equivalents of propyl tosylate at 65-67°C. The reaction is then cooled to room temperature, insoluble specieswere collected and washed with 8 volumes of MTBE.

G: 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole mixtures were dissolvedin 18 volumes of DMF and 1.25 equivalents of propyl tosylate at 65-67°C. The reaction is then cooled to room temperature, insoluble specieswere collected and washed with 8 volumes of MTBE.

H: 2,6 diamino-4,5,6,7-tetrahydro-benzothiazole mixtures were dissolvedin 10 volumes of DMF and 1.25 equivalents of propyl tosylate at 65-67°C. The reaction is then cooled to room temperature and insoluble specieswere collected. No washing step was performed.

Results are compiled in Table 2:

TABLE 2 Experiments for S_(N)2 preparation of pure enantiomers of2-amino- 4,5,6,7-tetrahydro-6-(propylamino)benzothiazole Ratio ofCondition G Condition H starting diamines Condition F (yield/ (yield/(6R):(6S) (yield/chiral purity) chiral purity) chiral purity) 80:20 —29%/99%   34%/98.2% 20:80 — 30%/99.4% 35%/95.7% 85:15 43%/86.8%36%/99.8% 39%/99.9% 15:85 52%/88.9% 27%/99.6% 37%/99.9% 90:10 47%/95.9%— — 10:90 58%/93.6% — — 95:5  50%/99.6% — —  5:95 47%/99.6% — —

The data in Table 2 demonstrates that both enantiomers of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole have similar, ifnot the same, solubility. Further, the data shows that the synthesis isequally efficient for either enantiomer of2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. These data alsodemonstrate that the enantiomers behave independently of one another, inthat the solubility of one enantiomer appears to be unaffected by theconcentration in solution of the other. For example, the varioussynthesis reactions carried out using condition F all have chemicalyields of about 50%, independent of the percentage of predominantdiamine enantiomer of the starting material. When the volume of theorganic solvent used in the synthesis reaction is increased, thechemical yield is reduced, but the chiral yield is increased. This isapparent by comparison of the reaction carried out in conditions F andG, where an 85:15 ratio of (6R):(6S) diamine produced a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product havingan 86.8% chiral purity for the (6R) enantiomer when the reaction used 10volumes of the organic solvent and a 99.8% chiral purity for the (6R)enantiomer when the reaction used 1.8 volumes of the organic solvent.Note also that the chemical yield was reduced in the reaction using alarger volume of organic solvent (43% yield in condition F and 36% yieldin condition G).

In Table 3, condition H is the same as condition F, except that therecovery step does not incorporate dilution in MTBE. The MTBE isobserved to increase2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole recovery (yield)from the synthesis reaction, but may reduce the overall chiral purity.This is born out by a comparison of the results for trials carried outin an 85:15 ratio of (6R):(6S) diamine, which produced a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole product having a86.8% chiral purity for the (6R) enantiomer when the reaction includedthe MTBE organic solvent and a 99.9% chiral purity for the (6R)enantiomer when the reaction did not include the MTBE organic solvent.The chemical yield was reduced by exclusion of the MTBE dilution in therecovery step; a 43% yield in condition C as opposed to a 39% yield incondition E.

Example 12 Conversion of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA saltto (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride

(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt(50 grams; 0.13 mol) was taken into 150 ml absolute ethanol and cooledto between 0 and 5° C. with continuous stirring. Concentrated HCl (33ml) was slowly added to the reaction while maintaining the temperatureat between 0 and 5° C., and the mixture was stirred for an additional 15minutes. MTBE (200 ml) was added to the mixture, and stirring wascontinued for an additional 1.5 hours at temperature. The reactionmixture was then filtered, washed twice with an MTBE/ethanol solution(2:1, 2×50 ml wash volumes), and dried under vacuum at 30° C. overnight.The final product was 34 grams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride, indicative of 92% yield, and a 97.3% chemical purity asdetermined by HPLC.

Example 13 Conversion of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA saltto (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride

(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt(10 grams; 0.026 mol) was dissolved in 200 ml IPAC and cooled to 15° C.with continuous stirring. HCl gas was bubbled into the slurry for 1hour. The mixture was then filtered, washed with IPAC, and driedovernight under vacuum at room temperature. The final product was 6.8grams of (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride, indicative of 92% yield, and a 97% chemical purity asdetermined by HPLC.

Example 14 Conversion of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA saltto (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine free base

(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt(25 grams; 0.065 mol) was dissolved in 200 ml DCM and mixed into aslurry. 10 ml of water was added and the mixture was basified with 12 mlof 6N NaOH to a pH of 11-12. The two phases were split, and the aqueouswas extracted with 200 ml of DCM. The combined organic phases were driedover MgSO₄, filtered over Celite® and concentrated. The residue wasdissolved in 100 ml MTBE and slurried for several hours. The solids werethen filtered, washed with MTBE and dried under vacuum at 35° C. Thefinal product was 9.1 grams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride, indicative a of 66% yield, and a 98% chemical purity asdetermined by HPLC.

Example 15 Conversion of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA saltto (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine free base

Freebase formation was performed on a 200 gram scale. A 5 L, threenecked, round-bottomed flask, equipped with an over head stirrer,thermometer, and addition funnel was charged with 200 g (0.522 mol) of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA saltand 1 L of water. The mixture was stirred and cooled to 10° C. Theslurry was basified to a pH of about 11-12 by the slow addition of 200ml of 6 N NaOH over a period of 15 min. The reaction mixture was dilutedwith 500 ml of brine (sodium chloride dissolved in water) and extractedwith 3×1 L of dichloromethane. The combined organic phases were washedwith 1.0 L of brine, dried over MgSO₄, filtered and concentrated todryness. The residue was triturated with 1 L of 1:1 IPAC:Heptane, theresulting slurry was stirred for 1 hour, filtered and the filter cakewas washed with 2×250 ml of 1:1 mixture of IPAC:Heptane. The filter cakewas collected and dried at 40° C. under high vacuum for 24 hours to give94.1 grams (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine(85.5%) as a white solid. The chemical purity was 100% AUC as tested byHPLC, and the chiral purity was 100% AUC as tested by HPLC. ¹H NMR and¹³C NMR was used to confirm the structure: ¹H NMR (300 MHz, DMSO-δ6) δ6.6 (s, 2H), 2.8 (m, 2H), 2.5 (m, 2H, merged with DMSO peak), 2.2 (m,1H), 1.9 (m, 1H), 1.5-1.3 (m, 4H), 0.85 (t, 3H); ¹³C NMR (300 MHz,DMSO-d6) δ 166.2, 144.8, 113.6, 54.2, 49.1, 30.0, 29.6, 25.2, 23.5,12.3.

Example 16 Conversion of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine free base to(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride

The freebase of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine (4.8 grams;0.022 mol) was dissolved in 200 ml of IPAC and cooled to 15° C. HCl gaswas bubbled into the slurry for 1 hour. The mixture was then filtered,washed with IPAC and dried under vacuum at room temperature overnight.The final product was 6.4 grams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride, indicative of 100% yield, and a 97% chemical purity asdetermined by HPLC.

Example 17 Conversion of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine free base to(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride

The freebase of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine (50 grams;0.13 mol) was dissolved in 500 ml of IPAC. Under continuous stirring,the mixture was slowly charged with 78 ml of concentrated HCl at atemperature of 25° C. The mixture was stirred overnight at ambientconditions (˜25° C.), filtered and dried under vacuum at 40° C. Thefinal product was 68 grams of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminedihydrochloride, indicative of 95% yield.

Example 18 Optical purification of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine usingachiral acid addition

2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole entantioenriched for the (6R) enantiomer (˜300 mg) was dissolved in 10 ml of thechosen solvent at 75° C. Complete dissolution was observed in allsamples. Acid addition was made at 1.05 molar equivalents for the p-TSA(solvent is ethanol; 2.97 ml of 0.5 M acid) and MSA (solvent isacetonitrile; 1.49 ml of 1.0 M acid), and 2.05 molar equivalents for thefumaric (solvent is acetonitrile; 5.84 ml of 0.5 M acid) and phosphoric(solvent is acetonitrile; 2.90 ml of 1.0 M acid). The reaction mixtureswere cooled to room temperature at a rate of 25° C./hour and stirred atroom temperature for an additional 19 hours.

The products of purification processes were then analyzed by HPLC forchemical and chiral purity. FIG. 3A shows an exemplary HPLC tracestarting material. In FIG. 4A, a large(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine peak can beobserved at about 6 minutes and a much smaller(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine peak can beseen at about 9 minutes. The area of these peaks provides an estimatedcomposition for the mixture which is shown in the table below the trace,and shows the mixture as containing about 902% (6R) and 8.8%(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. FIG. 3Bshows an exemplary trace of the2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole productfollowing purification. In FIG. 3B, a large(6R)₂-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole peak isobserved at about 6 minutes, and no(6S)2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole peak isobserved. It is additionally of note that several other minor peaks arealso reduced or eliminated in the product trace, and the table below thetrace indicates that(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine makes up99.9% of the product solution which is within the limits of analyticaldetectability. Results for each exemplary reaction are provided in Table3:

TABLE 3 Experiments for preparation of the(6R)-4,5,6,7-tetrahydro-N6-propyl- 2,6-benzothiazole-diamine Salt/AcidSolvent Batch Size Results p-TSA ethanol 298.7 mg Yield = 489.5 mg(90.3%) Start Chiral Purity = 91% AUC (6R) by HPLC Final Chiral Purity =100% AUC by HPLC MSA acetonitrile 300.0 mg Yield = 431.8 mg (98.9%)Start Chiral Purity = 91% AUC (6R) by HPLC Final Chiral Purity = 99.23%AUC by HPLC fumaric acetonitrile 301.0 mg Yield = 532 mg (84.2%) (hotethanol) Start Chiral Purity = 91% AUC (6R) by HPLC Final Chiral Purity= 99.26% AUC by HPLC phosphoric acetonitrile 299.4 mg Yield = 592 mg(~100%) Start Chiral Purity = 91% AUC (6R) by HPLC Final Chiral Purity =100% AUC by HPLC

The solids obtained by this trituration step were isolated by filtrationand dried under high vacuum at room temperature. These products wereanalyzed by HPLC, NMR, thermal gravimetric analysis, differentialscanning calorimetry, X-ray powder diffraction (XPRD), Fourier transforminfrared spectroscopy and moister-sorption analysis. The XPRD patternsshowed that the p-TSA, MSA and fumarate salt forms of the(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine werecrystalline, while the phosphate salt form of the(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine wasamorphous.

Example 19 Industrial Scale Resolution of Racemic Diamine

A 72 L, unjacketed reactor was charged with racemic 2,6diamino-4,5,6,7-tetrahydro-benzothiazole (4.5 kg; 26.6 mol) and 58.5 Lwater, and heated as a suspension to a temperature of about 60° C. to65° C. Resolution of the enantiomers was achieved by addition of oneequivalent of (D)-(−)-Tartaric acid (3991 grams; 26.6 mol) in 4.5 L ofwater, after which the resulting solution was heated to a temperature ofabout 70° C. to 75° C. and maintained at this temperature for about 1hour. The mixture was allowed to cool to a temperature of about 20° C.to 25° C. and stirred for an additional 15 hours, after which themixture was filtered and the solids were washed 3× with water (6.3 Leach wash).

The wet solids, which contain the (6R) enantiomer of the diamine, werecharged to the reactor followed by 54 L of water, and the mixture washeated to a temperature of about 70° C. to 75° C. for 2 hours. Themixture was allowed to cool to a temperature of about 20° C. to 25° C.and stirred for 17 hours. The mixture was then filtered and the solidswere washed 2× with water (4.5 L each wash). The wet solids weretransferred to a jacketed reactor and the reactor was charged with 8.1 Lof water. The mixture was cooled to a temperature of about 0° C. to 5°C. and cautiously charged with concentrated 1.6 L of HCl, followed by1.2 L of 50% NaOH to achieve a pH of about 9-10. During the addition,the temperature was maintained at about 0° C. to 5° C., and stirred foran additional hour at temperature. The resulting mixture was thenfiltered and the solids were washed 2× with cold (0° C. to 5° C.) water(1.1 L each wash). The solids were transferred to a jacketed reactor andwere reslurried once more with 4.5 L of water at 0° C. to 5° C. Thesolids were filtered and dried under warm air (40° C. to 45° C.) to give1940 grams of the product ((6R) diamine) as a white solid, with an 86%yield for the (6R) enantiomer.

The mother liquors of the initial resolution step, which contain the(6S) enantiomer of the diamine, were concentrated to afford diamine witha 95.5% yield for the (6S) enantiomer. Results of reactions performedusing 1000, 4500 and 41.00 grams of starting material are provided inTable 4

TABLE 4 Experiments for industrial scale resolution of the (6R)enantiomer of diamine Input Yield (%) of Chemical Purity Chiral Purity(grams) (6R) enantiomer (AUC % by HPLC) (AUC % by HPLC) 1000 76 >99 98.34500 86 >99 98.5 4100 54 >99 98.5

Example 20 Industrial Scale Preparation of Propyl Tosylate

A 100 L glass, jacketed reactor was charged with 1-propanol (2.098 kg;34.9 mol), triethylamine (4.585 kg; 45.3 mol; 1.3 equivalents) and DCM(20.1 L). The mixture was cooled to a temperature of about 5° C. to 15°C. and cautiously charged with a solution of p-toluenesulfonyl chloride(6 kg; 31.47 mol; 0.9 equivalents) in DCM (10.5 L) over 30 minutes. Oncethe addition was complete, the mixture was warmed to a temperature ofabout 18° C. to 22° C. and stirred for 12 hours. The reaction mixturewas assayed by ¹H NMR (in CDCl₃) and deemed complete. HCl (6 N; 2.98 L)was cautiously charged while maintaining the temperature below 25° C.The aqueous phase was removed, and the organic phase was washed 2× withwater (21 L each wash), dried with MgSO₄, and filtered over Celite®. Thefiltered solids were then washed with DCM (4 L) and concentrated to aresidue. The residue was dissolved in heptane and concentrated again toafford a final propyl tosylate product (6.385 kg, 95% yield).

Example 21 Industrial scale preparation of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine p-TSA salt:Condition C

A 72 liter unjacketed reactor was charged with 1.84 kg (10.87 mol) of(6R)-2,6 diamino-4,5,6,7-tetrahydro-benzothiazole ((6R) diamine),followed by 14.7 L of dimethyl formamide (DMF). Under continuousstirring, the mixture was heated to a temperature of between 65° C. and68° C. A solution of 2926 grams propyl tosylate and 1761 gramsdiisopropylethyleamine in 3.5 L DMF was added slowly over a period of 2hours. The reaction was continued at 67° C. for an additional 4 hours,after which the solution was gradually cooled to room temperature (18°C. to 22° C.) and stirred for an additional 15 hours. The solution wasdiluted with 14.7 L of MTBE over a time period of 30 minutes, andstirred for an additional 1 hour. The precipitated material wascollected by filtration and washed with 7.3 L MTBE, followed by 3 washesof 3.7 L each of ethanol, and a wash with 9.2 L heptane. The washedprecipitated cake was dried under high vacuum at 30° C. to 35° C. Thefinal weight of the dried product was 2090 grams, representing a 50%yield.

Example 22 Purity of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine

The Limit of Quantitation (LOQ) to 0.05% precision was determined fromsix replicate preparations of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine. The RSD wasmeasured to be 6.3% and the S/N (signal to noise ratio) was measured tobe 61:1. The pre-defined acceptance criteria at LOQ was to have aS/N>10:1 and for the RSD to be <20.0%. These results exceeded theacceptance criteria by a wide margin.

Signal-to-noise levels can vary for numerous reasons, including pumpbehavior, air in lines, extent of mobile phase degassing,system-to-system variations and electronic fluctuations. The LOD hadpreviously been estimated to be 0.03% based upon the 0.1% preparationwhich produced a S/N of 30:1. Although the recent 0.05% levels produceda S/N of 61:1, based upon the overall history of the method, theestimated LOD will remain at the stated 0.03%.

Linearity was determined over the range 0.05%-150% of nominal (0.2μg/mL-600 μg/mL). The correlation coefficient over this expanded rangewas determined to be 0.9999. This exceeded the pre-defined linearityacceptance criterion of >0.995.

The weight percent assay is on an as-is basis versus the currentstandard which has a purity of 94.0%. Each sample was prepared induplicate with single injections. Purity data is provided in the Table5:

TABLE 5 (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diaminePurity Data % Sample % area ave wt/wt ave Sample 118 100.0 95.1 Sample118 100.0 100.0 95.0 95.1 Sample 105 100.0 94.7 Sample 105 100.0 100.094.7 94.7 Sample 061 100.0 94.5 Sample 061 100.0 100.0 94.6 94.5 Sample326A 100.0 95.0 Sample 326A 100.0 100.0 94.7 94.8

Example chromatographs are provided in FIG. 4A-D. Specifically, FIG. 4Ais an HPLC Chromatograph of Sample 118; FIG. 4B is an HPLC Chromatographof Sample 105; FIG. 4C is an HPLC Chromatograph of Sample 061; and FIG.4 D is an HPLC Chromatograph of Sample 326A. These data show preparationof (6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine at 100%purity.

Example 23 Propyl tosylate concentration of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine

A solid phase extraction (SPE) procedure was developed to remove thehigh levels of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine prior toanalysis while maintaining recovery of propyl tosylate spiked at 1.5ppm. The SPE cartridges (Supelco Discovery DSC-18, 6 mL, 1 g) arepre-activated and washed with 6 mL acetonitrile (MeCN) followed by 6 mLof water. Five milliliters of 100 mg/mL solutions of KNS-760704 preparedin 5:95, MeCN/water with 0.5% phosphoric acid are then introduced intothe SPE cartridges. The acid sufficiently maintains the polarity of the(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine so that itcan be readily washed from the SPE cartridge with an additional 5 mL of5:95, MeCN/water with 0.5% phosphoric acid while retaining any propyltosylate. Any propyl tosyate is then eluted from the SPE cartridgesusing 5 mL of 95:5 MeCN/water. Fortunately, due to the sensitivitygained from method development experiments, no further sample enrichmentis needed and the samples are analyzed as-is. The samples are comparedto a standard of 1.5 ppm (0.15 μg/mL) propyl tosylate prepared in 95:5MeCN/water.

An exemplary UV spectrum of propyl tosylate eluted from the SPEcartridges is shown in FIG. 5.

Exemplary HPLC data from of the(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine eluted fromthe SPE cartridge is provided in FIGS. 6A and 6B. FIG. 6A shows an HPLCchromatograph of a propyl tosylate standard prepared from 100 mg ofpropyl tosylate. FIG. 6B shows an HPLC chromatograph of a 1 g sample of(6R)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine prepared bythe method of embodiments of the invention. No propyl tosylate isevident based on the data provided in FIG. 6B as indicated by theabsence of a peak corresponding to propyl tosylate (right).

Example 24 Chiral purity of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine

The chirality of the(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine prepared asdescribed above was tested using a Chiralpak IA column under HPLCconditions. About 25 mg of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine were appliedto the column. Exemplary HPLC traces of a(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine standard areprovided in FIG. 7A and an HPLC trace of a sample(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole-diamine is providedin FIG. 7B. Test data is provided in Table 6.

TABLE 6 % Recovery of(6S)-4,5,6,7-tetrahydro-N6-propyl-2,6-benzothiazole- diamine LevelPreparation % Recovery 100% 1 91.13 2 91.18 3 97.61 Average 93.3 % RSD4.0

Although the present invention has been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontained within this specification.

1. A process for preparing a chirally purified substituted4,5,6,7,-tetrahydro-benzothaizole diamine comprising: heating a solutioncomprising entantiomerically enriched 4,5,6,7-tetrahydro-benzothiazolediamine of general formula (I):

wherein: R₁ represents a hydrogen atom, an alkyl group having 1 to 6carbon atoms, an alkenyl or alkynyl group each having 3 to 6 carbonatoms, an alkanoyl group having 1 to 6 carbon atoms, a phenyl alkyl orphenyl alkanoyl group having 1 to 3 carbon atoms in the alkyl part,whilst the above-mentioned phenyl nuclei may be substituted by 1 or 2halogen atoms; R₂ represents a hydrogen atom or an alkyl group with 1 to4 carbon atoms; R₃ represents a hydrogen atom, an alkyl group with 1 to7 carbon atoms, a cycloalkyl group having 3 to 7 carbon atoms, analkenyl or alkynyl group having 3 to 6 carbon atoms, an alkanoyl grouphaving 1 to 7 carbon atoms, a phenyl alkyl or phenyl alkanoyl grouphaving 1 to 3 carbon atoms in the alkyl part, whilst the phenyl nucleusmay be substituted by fluorine, chlorine or bromine atoms, R₄ representsa hydrogen atom, an alkyl group with 1 to 4 carbon atoms, an alkenyl oralkynyl group having 3 to 6 carbon atoms; and at least one or R₁, R₂, R₃or R₄ is a hydrogen in an organic solvent; and an alkyl sulfonate or analkyl halide in a solvent to form a reaction mixture; reacting thereaction mixture; and recovering a chirally purified substituted4,5,6,7-tetrahydro-benzothiazole diamine. 2-28. (canceled)
 29. Chirallypurified 2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazoleprepared by a process comprising: heating a solution comprisingentantiomerically enriched 2,6 diamino-4,5,6,7-tetrahydro-benzothiazoleand a propyl halide or a propyl sulfonate to form a reaction mixture;reacting the reaction mixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. 30-52.(canceled)
 53. A process for preparing a chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole comprising:heating a solution comprising 2,6diamino-4,5,6,7-tetrahydro-benzothiazole in an organic solvent; addingto the heated solution propyl sulfonate or a propyl halide to form areaction mixture; and reacting the reaction mixture for up to about 12hours. 54-79. (canceled)
 80. A process for preparing chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole comprising:heating a solution comprising 2,6diamino-4,5,6,7-tetrahydro-benzothiazole; adding a propyl halide or apropyl sulfonate to the heated solution slowly over from about 0.5 hoursto about 2 hours to form a reaction mixture; reacting the reactionmixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. 81-101.(canceled)
 102. Chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole prepared by aprocess comprising: heating a solution comprising 2,6diamino-4,5,6,7-tetrahydro-benzothiazole; adding a propyl halide or apropyl sulfonate to the heated solution slowly over from about 0.5 hoursto about 2 hours to form a reaction mixture; reacting the reactionmixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. 103-125.(canceled)
 126. A process for preparing chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole comprising:dissolving entantiomerically enriched2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in an organicsolvent to form a solution; heating the solution to from about 50° C. toabout 125° C.; adding an acid to the solution to form a reactionmixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. 127-128.(canceled)
 129. A chirally pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole prepared by aprocess of claim
 126. 130. A process for preparing chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole comprising:dissolving entantiomerically enriched2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole in an organicsolvent to form a solution; heating the solution to from about 50° C. toabout 125° C.; adding an achiral salt to the solution to form a reactionmixture; and recovering the chirally purified2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole. 131-132.(canceled)
 133. A chirally pure2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole prepared by aprocess of claim
 130. 134. A process for preparing a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole dihydrochloridecomprising: dissolving a2-amino-4,5,6,7-tetrahydro-6-(propylamino)benzothiazole salt in anorganic solvent to form a solution; cooling the solution to atemperature of from about 0° C. to about 5° C.; adding concentrated HCland an organic solvent to the cooled solution; and stirring the solutionat a temperature of about 0° C. to about 5° C.